A Dissertation

entitled

Preclinical evaluation of a potential treatment for ADHD targeting the serotonin 1B

receptor subtype

by

Yasir Hazim Saber

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Doctor of Philosophy Degree in Experimental Therapeutics

______Dr. F. Scott Hall, Committee Chair

______Dr. Youssef Sari, Committee Member

______Dr. Frederick Williams, Committee Member

______Dr. Zahoor Shah, Committee Member

______Dr. Cyndee Gruden, Dean College of Graduate Studies

The University of Toledo

December 2019

Copyright 2019, Yasir Hazim Saber

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author.

An Abstract of

Preclinical Evaluation of a Potential Treatment for ADHD Targeting The Serotonin 1B Receptor Subtype

by

Yasir Hazim Saber

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Experimental therapeutics

The University of Toledo December 2019

Perturbations in dopamine signaling, and consequent alterations in corticostriatal function, have been implicated in attention deficit/ hyperactivity disorder (ADHD).

Paradoxically, the primary treatments for ADHD are monoamine uptake inhibitors, which are psychostimulant-like drugs. However, these drugs work in only a proportion of individuals with ADHD and other concerns limit their effective use. Current non- stimulant ADHD medications also have shortcomings. A crucial element of identification novel drugs treatments are valid animal models of psychiatric disorders. The experiments presented here further validated the dopamine transporter knockout (DAT KO) mouse model and then used this model to evaluate a potential new approach to the treatment of

ADHD, via inhibition of the serotonin 1B receptor subtype (5HT1B). Substantial evidence has shown that DAT -/- mice exhibit ADHD-like deficits that are ameliorated by treatment with drugs that are effective ADHD therapies. However, since these mice show features that are not like ADHD, DAT +/- mice were also investigated.

Additionally, sex has been an underexplored feature of this model and ADHD overall, in

iii part because the presentation of ADHD symptoms is different in females. The results presented here found additional evidence that DAT -/- mice show ADHD-like phenotypes that can be reversed by effective ADHD treatments. Moreover, DAT +/- mice also exhibited ADHD-like phenotypes, although less severe than DAT -/- mice, and these deficits could be reduced by effective ADHD treatments as well. Both the deficits and responses to effective ADHD treatments were sex dependent – both males and females showed similar deficits, but males were more effected in some symptom realms and females in others. Finally, the 5HT1B receptor antagonist SB 224289 reduced many of the deficits in both DAT –/- and DAT +/- mice, and importantly were shown to reduce impulsivity in the 5-Choice Continuous Performance Test in DAT +/- mice. In summary the data in this thesis provide additional evidence for the validity of the DAT KO model, and in particular the DAT +/- model, as well as evidence supporting the potential of

5HT1B antagonism as a potential new, non-stimulant approach to the treatment of

ADHD.

iv Acknowledgements

First and foremost, I would like to thank the Almighty Allah for his grace and guidance throughout all of my life’s journey. Next, I would like to express my deepest gratitude to my PhD advisor, Dr. F. Scott Hall for his unlimited support and guidance throughout my PhD journey. I have been so lucky to be in his lab. I will try my best to follow his example as person and a mentor. I would never have reached this landmark without Dr. Hall’s patience, motivation and continuous belief in me. I would also like to thank my committee members: Dr. Frederick E. Williams, Dr. Youssef Sari, and Dr.

Zahoor Shah for their support and for asking challenging questions that pushed me to broaden my knowledge. I want to thank my graduate representative, Dr. Sarver for his support. In addition, I would like to thank my awesome lab members: Chen Yu, Huyen T.

Tran, Dawn Muskiewicz, Nicole Frommann, and Alexander Wisner for their help and support. I would like to thank Dr. Jeffrey Sarver, Dr. Caren Steinmiller and Dr. Jill

Trendel for their support and advice. Last but not least, I would thank my spouse Zaharaa

Anaz for her support and patience and my kids Abdulazeez, Moataz and Yamn for being just wonderful; my father, Hazim Anaz, and my mother, Hanan Ahmed, for their encouragement, support, and source of joy. I am very thankful for Friends (Qasim

Alhadidi, Ehsan Sabar, Salah Alaqel, Mohamed Bin-said, and Abdul Saheb) and my brothers, Dr. Ammar and Dr. Aws, and my sister, Stabraq.

v

Table of Contents

Abstract ...... iii

Acknowledgements ...... v

Table of Contents ...... vi

List of Figures ...... Error! Bookmark not defined.i

1 Introduction ...... 1

References ...... 10

2 Evaluation of the Validity of Animal models of Attention Deficit Hyperactivity

Disorder (ADHD) ...... 17

1.0 Introduction ...... 19

2.0 Animal models using inbred rodent strains ...... 23

2.1 The spontaneously hypertensive rat (SHR) ...... 23

2.2 Selectively bred hyperactive mice ...... 26

3.0 Genetically-modified rodent models ...... 27

3.1 Genetic modifications targeting the dopamine transporter ...... 27

3.1.1 Dopamine transporter knock out (DAT KO) strains ...... 29

3.1.2 Heterozygous DAT KO mice (DAT +/- mice) ...... 40

3.1.3 Other genetic modifications targeting DAT ...... 44

3.2.0 Genetic modifications targeting other genes in rodent models ...... 48

3.2.1 Neurokinin 1 receptor KO (NK1R KO) mouse ...... 49

vi 3.2.2 Coloboma (SNAP25 knockout) mouse ...... 53

. 3.2.3 Mutant Thyroid Hormone Receptor β Knock-in (TRβPV KI)

mouse ...... 55

3.2.4 Adhesion G-protein coupled receptor L3 KO (ADGRL3 KO)

mice ...... 58

3.2.5 P35 (cyclin-dependent kinase 5 cofactor p35) KO mice ...... 60

3.2.6 ADF actin depolymerization factor (ADF)/cofilin double

mutant mice ...... 61

3.2.7 Guanylyl Cyclase-C 3 KO (GC-C KO) mice ...... 63

3.2.8 Phosphoinositide 3 kinase γ KO (PI3Kγ KO) mouse ...... 64

3.2.9 39,XY*O mouse / steroid sulfatase (STS) KO mice ...... 66

3.2.10 γ aminobutyric acid transporter 1 (GAT1) KO mice ...... 67

3.2.11 Genetic modifications of nicotinic acetylcholine receptors

(nAChRs) ...... 68

3.2.12 Fragile X mental retardation 1 (FXMR1) KO mice...... 69

4.0 Conclusion ...... 70

References ...... 73

3 The effects of reduced dopamine transporter expression on the sex-dependent

effects of isolation-rearing ...... 115

1.0 Introduction ...... 118

2.0 Materials and methods ...... 122

2.1 Subjects ...... 122

2.2 Rearing Conditions ...... 123

vii 2.3 Locomotor activity ...... 123

2.4 Prepulse inhibition (PPI) ...... 124

2.5 Cliff Avoidance Reaction (CAR)...... 124

2.6 Statistical Analysis ...... 125

3.0 Results ...... 126

3.1 Locomotor activity ...... 126

3.2 Cliff Avoidance Reaction (CAR)...... 133

3.3 Pre-pulse Inhibition of Acoustic Startle (PPI) ...... 135

4.0 Discussion ...... 137

References ...... 142

4 Sex-dependent effects of the serotonin 1B receptor antagonist SB 224289 in an

animal model of Attention Deficit Hyperactivity Disorder ...... 150

1.0 Introduction ...... 153

2.0 Materials and methods ...... 158

2.1 Subjects ...... 158

2.2 Locomotor activity ...... 159

2.3 Prepulse inhibition (PPI) ...... 159

2.4 Cliff avoidance reaction (CAR) ...... 160

2.5 Drug Treatments ...... 161

2.6 Statistical Analysis ...... 161

3.0 Results ...... 162

3.1 Locomotor activity ...... 162

3.2 PPI ...... 167

viii 3.3 CAR ...... 169

4.0 Discussion ...... 172

References ...... 178

5 Preclinical assessment of the Serotonin 1B receptor as a novel target for the

treatment of Attention Deficit Hyperactivity Disorder ...... 189

1.0 Introduction ...... 192

2.0 Materials and methods ...... 195

2.1 Subjects ...... 195

2.2 Locomotor activity ...... 196

2.3 Cliff Avoidance Reaction (CAR)...... 196

2.4 Drug Treatments ...... 197

2.5 Statistics and data analysis ...... 198

3.0 Results ...... 199

3.1 Effects of Atomoxetine on locomotor activity in DAT +/+,

DAT +/-, and DAT -/- mice ...... 199

3.2 Effects of SB 224289 of locomotor activity in DAT +/+, DAT

+/-, and DAT -/- mice ...... 201

3.3 Cliff Avoidance Reaction (CAR) deficits in DAT KO mice .204

3.4 Effects of Atomoxetine (ATX) on CAR in DAT KO mice ...208

3.5 Effects of SB 224289 on CAR in DAT KO mice ...... 210

4.0 Discussion ...... 211

References ...... 217

ix 6 Heterozygous dopamine transporter knockout mice as an animal model of ADHD:

effects of amphetamine and the serotonin 1B receptor antagonist SB224289 ....229

1.0 Introduction ...... 232

2.0 Methods and materials ...... 238

2.1 Subjects ...... 238

2.2 Apparatus ...... 239

2.3 Training protocol: 5-Choice Continuous Performance Test

(5-CCPT) ...... 239

2.4 5-CCPT Challenge sessions ...... 241

2.5 Drug Treatments ...... 242

2.6 Statistics and data analysis ...... 243

3.0 Results ...... 244

3.1 Amphetamine effects on behavioral performance in the 5-CCPT .....244

3.2 Effects of SB 224289 on behavioral performance in the 5-CCPT.....253

4.0 Discussion ...... 258

4.1 DAT +/- mice as a model of ADHD ...... 258

4.2 Predictive Validity of the DAT +/- model ...... 263

4.3 SB 224289 as a potential therapeutic for ADHD ...... 264

Conclusions ...... 265

References ...... 266

7 Summary: Preclinical evaluation of a potential treatment for ADHD targeting the

serotonin 1B receptor subtype ...... 283

1.0 Introduction ...... 284

x 2.0 The DAT KO model of ADHD: Isolation rearing ...... 288

3.0 The DAT KO model of ADHD: Sex and the effects of partial DAT deletion ...... 289

4.0 5HT1B Antagonism as a potential treatment for ADHD...... 292

5.0 Final Conclusions...... 293

References ...... 294

xi List of Figures

2-1 Schematic illustration of the neural circuitry that may underlie the effects of

nisoxetine, methylphenidate, and nicotine in DAT KO mice involving the medial

prefrontal cortex (mPFC)...... 35

3-1 Locomotion in male DAT+/+, DAT+/-, socially-reared and isolation reared mice

...... 127

3-2 Locomotor activity in male DAT+/+ and DAT+/- ...... 128

3-3 Locomotion in female DAT+/+, DAT+/-, socially reared and isolation-reared

mice habituation saline and amphetamine ...... 129

3-4 Locomotor activity of different DAT genotype female mice ...... 130

3-5 % CAR success in male for socially-reared mice and isolation reared mice ...... 132

3-6 % CAR success in female for socially-reared mice and isolation reared mice ...133

3-7 % CAR success comparisons of male and female mice ...... 135

3-8 Rearing condition and DAT genotypes on prepulse inhibition ...... 136

4-1 Schematic illustration of the neural circuitry that may underlie the effects of

nisoxetine, methylphenidate, and nicotine in DAT KO mice involving the medial

prefrontal cortex (mPFC)...... 157

4-2 Locomotor activity in a novel environment in DAT+/+, DAT +/- mice ...... 163

4-3 The effects of SB224289 on locomotor activity on male and female...... 164

4-4 PPI in male and female DAT+/+, DAT+/- and DAT-/-...... 166

4-5 The effect of SB224289 on PPI in male and female DAT+/+, DAT+/-, and DAT -

/-mice of prepulse intensity ...... 168

xii 4-6 % CAR in male and female, DAT+/+, DAT+/-, and DAT-/- mice ...... 170

4-7 % CAR in male and female, DAT+/+, DAT+/- and DAT-/- mice ...... 171

5-1 Locomotor activity in male DAT+/+, DAT+/- and DAT-/-after administration of

saline or 1, 2 and 10 mg/kg ATX ...... 199

5-2 Locomotor activity in male DAT+/+, DAT+/- and DAT-/-after administration of

saline or 1, 2 and 10 mg/kg ATX ...... 200

5-3 Locomotor activity in male DAT+/+, DAT+/- and DAT-/- mice administration of

saline 10, 20, 30 mg/kg SB224289 ...... 202

5-4 Locomotor activity in female DAT+/+, DAT+/- and DAT-/- mice administration

of saline 10, 20, 30 mg/kg SB224289 ...... 203

5-5 Effects of sex on CAR ...... 204

5-6 Genotypic comparisons between male and female, DAT+/+, DAT+/-, and DAT-/-

mice in the CAR test ...... 205

5-7 Effects of atomoxetine in male DAT +/+, DAT+/-, and DAT-/- mice in CAR...206

5-8 Effects of atomoxetine in female DAT +/+, DAT+/-, and DAT-/- mice in CAR

...... 207

5-9 Effects of SB224289 in male DAT +/+, DAT+/-, and DAT-/- mice in CAR .....208

5-10 Effects of SB224289 in female DAT +/+, DAT+/-, and DAT-/- mice in CAR ..209

6-1 the effects of amphetamine (saline, 0.3, 0.66, 1.5 mg/kg IP) on accuracy in

DAT+/+ and DAT+/- mice at ITI 4s and ITI 8s and SD 2 s, SD1 s and SD 0.05

...... 245

xiii 6-2 The effects of amphetamine (saline, 0.3, 0.66, 1.5 mg/kg IP) on correct % in

DAT+/+ and DAT+/- mice at ITI 4s and ITI 8s and SD 2 s, SD1 s and SD 0.05

...... 246

6-3 The effects of amphetamine (saline, 0.3, 0.66, 1.5 mg/kg IP) on incorrect % in

DAT+/+ and DAT+/- mice at ITI 4s and ITI 8s and SD 2 s, SD1 s and SD 0.05

...... 247

6-4 Incorrect latency distribution difference between DAT+/+ and DAT+/- male mice

and the effect of amphetamine ...... 248

6-5 The effects of amphetamine (saline, 0.3, 0.66, 1.5 mg/kg IP) on % premature and

% perseveration in DAT+/+ and DAT+/- mice ...... 249

6-6 The effects of amphetamine (saline, 0.3, 0.66, 1.5 mg/kg IP) on % omission in

DAT+/+ and DAT+/- mice at ITI 4s and ITI 8s and SD 2 s, SD1 s and SD 0.05

...... 250

6-7 The effects of amphetamine (saline, 0.3, 0.66, 1.5 mg/kg IP) on the sensitivity

index and the response index in DAT+/+ and DAT+/- mice at ITI4 and ITI 8 ...251

6-8 The effects of SB224289 (vehicle, 10, 20, and 30 mg/kg IP) on accuracy in

DAT+/+ and DAT+/- mice at ITI 4s and ITI 8s and SD 2 s, SD1 s and SD 0.05

...... 252

6-9 The effects of SB224289 (vehicle, 10, 20, and 30 mg/kg IP) on correct % in

DAT+/+ and DAT+/- mice at ITI 4s and ITI 8s and SD 2 s, SD1 s and SD 0.05

...... 254

xiv 6-10 The effects of SB224289 (vehicle, 10, 20, and 30 mg/kg IP) on incorrect % in

DAT+/+ and DAT+/- mice at ITI 4s and ITI 8s and SD 2 s, SD1 s and SD 0.05

...... 255

6-11 The effects of SB224289 (vehicle, 10, 20, and 30 mg/kg IP) on sensitivity index,

premature response and response index in DAT+/+ and DAT+/- male mice at ITI

4 s and ITI 8 s...... 256

6-12 The effects of SB224289 (vehicle, 10, 20, and 30 mg/kg IP) on % omission in

DAT+/+ and DAT+/- mice at ITI 4s and ITI 8s and SD 2 s, SD1 s and SD 0.05

...... 257

xv Chapter 1

Introduction: Preclinical evaluation of a potential treatment for ADHD targeting the serotonin 1B receptor subtype

Yasir H. Saber a, b a Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH, USA; b Ninevah College of medicine, Ninevah university, Mosul, Iraq

1

Attention deficit/hyperactivity disorder (ADHD) is one of the most prevalent developmental neuropsychological disorders in children. ADHD has been reported to affect between 5.3 and 11% of the school-age children (Polanczyk et al., 2014; Visser et al., 2014) and 3.4% of the adult population (Fayyad et al., 2007). ADHD diagnoses have continued to increase over the last decade, especially among adults and females (Fairman et al., 2017). The extent to which this reflects a true increase in incidence, or better diagnosis is unclear. The symptoms of ADHD differ somewhat as a factor of sex and age, and this has affected rates of diagnosis, with some groups being underdiagnosed. This may also mean that different treatments may by necessity differ among patients, based on their symptom profiles. Treatment is need for ADHD because it has a negative impact on academic achievement, social interactions, and accomplishment later in life. Indeed, although the symptoms are less severe and debilitating than many other psychiatric disorders, ADHD still represents a substantial emotional and financial burden for families and communities. In addition, there is a high comorbidity of ADHD with other psychiatric disorders, and especially concerning is a tendency for increased rates of drug use and drug addiction in adolescents with ADHD, which may result from some of the core symptoms of ADHD or perhaps even results from early exposure to psychostimulant drugs as a part of ADHD therapies. ADHD core symptoms include attentional impairment, hyperactivity, and impulsivity, but deficits in executive function and other cognitive impairments should also be included. ADHD has been divided into subtypes based on the predominate symptoms: attentional deficit with no hyperactivity, hyperactivity, impulsivity with no attentional deficit, or combined attentional deficit and hyperactivity (DSM-V; American Psychiatric Association (2013), but it is perhaps better 2

to think of the wider profile of ADHD deficits as independent symptoms that may or may not present in any particular patient. Although many non-pharmacological approaches are taken to the management of ADHD symptoms, 69% of children with ADHD are prescribed medication, with psychostimulant drugs like methylphenidate and d- amphetamine (Ritalin and Adderall, respectively) being the most commonly prescribed drugs (Punja et al., 2016; Storebo et al., 2015). Psychostimulant use has been suggested to increase the potential for the development of drug addiction and raised concern regarding the use of these drugs on the development of dopaminergic systems when given at an early age in children with ADHD (Gerlach et al., 2013). Although some non- psychostimulant medications like atomoxetine and clonidine are available for the treatment of ADHD, a relatively high percentage (35%) of ADHD patients do not respond well to these medications (Beherec et al., 2014), and side effects may limit the ability to reach effective therapeutic doses. Accordingly, the development of a novel non- stimulant medication with a more favorable side-effect profile is highly desirable. It should be possible to accomplish this goal by finding a druggable alternative pathway to psychostimulants or atomoxetine to treat or alleviate ADHD symptoms. Of course, one of the difficulties in identifying such an alternative pathway is the limited understanding that we currently have of the way in which even the oldest ADHD treatments, psychostimulant drugs, produce their effects (Rubia et al., 2014).

Recent work with a new animal model of ADHD, the DAT KO mouse, has suggested how psychostimulant drugs may actually work to modulate corticostriatal neurocircuitry, and based upon this model alternative treatment approaches may be posited. The findings, discussed in detail below, suggest that psychostimulant ADHD 3

medications act in ADHD by blocking norepinephrine transporters in the prefrontal cortex. Moreover, this model also suggests that manipulations of cholinergic or serotonergic receptors within this circuit may also have therapeutic effects in ADHD.

Preclinical studies are indispensable methods for developing medications for psychiatric disorders for which the primary dysfunctions and therapeutic outcomes are behavioral in nature. Although other animal models of ADHD have been proposed, they all have been substantial short-comings (Arime et al., 2011; Fan et al., 2012). The validity of different animal models is discussed in Chapter 2 of this thesis. As that extensive analysis shows, the majority of models have had numerous shortcomings. Indeed, most proposed ADHD models have focused largely upon hyperactivity as an operational measure, rather than upon cognitive or attentional deficits. This is problematic for two reasons. Firstly, many

ADHD patients do not present with hyperactivity, particularly female and adult patients.

Secondly, although such models may effectively identify psychostimulant-like drugs that reduce hyperactivity, they would be expected to be less effective in identifying other novel non-stimulant pharmacological effects, and particular drugs that may be more affective at treating cognitive or attentional deficits in ADHD if those deficits are not shown in the model. The weaknesses of these model have resulted, at least in part, from limited understanding of the causes of ADHD in humans that can be incorporated into animal models. Consequently, these previous models have behavioral and physiological phenotypes not related to ADHD.

As clearly shown in the analysis in Chapter 2, dopamine transporter knockout

(DAT KO) mice provide a much more valid model of ADHD, which included a wider range of ADHD-like symptoms than many other models, demonstration of the 4

effectiveness of a range of ADHD treatments on a range of ADHD-like behavioral deficits, and some similar underlying changes in brain function to what is observed in

ADHD patients. However, some of these criticisms about differences in underlying biology from ADHD and the presence of non-ADHD-like phenotypes can also be applied to mice with complete DAT deletion (DAT -/-) mice. The earliest observed phenotype in these mice was locomotor hyperactivity, and DAT -/- mice also exhibit some impairments not characteristic of ADHD. However, in other respects, this model is certainly better than previous models. Dopamine and DAT certainly attract special attention in the investigation of the pathophysiology of ADHD, as well as medications development, in part because the first-line treatments for ADHD are psychostimulant drugs (Gold et al., 2014; Gowrishankar et al., 2014). DAT -/- mice are hyperactive and show reduced habituation to environments and other stimuli (Giros et al., 1996; Sora et al., 2001; Sora et al., 1998). Moreover, this hyperactivity is attenuated by psychostimulant drugs, including those that are prescribed for ADHD (Gainetdinov et al.,

1999). That study also established the first connection between serotonin and ADHD in the DAT KO model, although the initial evidence may be artifactual, the result of increased tendencies to develop the serotonin behavioral syndrome in those mice (Fox et al., 2013). Furthermore, evidence has accumulated that blockade of the norepinephrine transporter (NET) mediates the reversal of DAT KO-induced impairments by psychostimulant drugs (Arime et al., 2012; Yamashita et al., 2006), and by implication may mediate the therapeutic effects of these drugs in humans. For example, DAT-/- mice have impairments in prepulse inhibition of acoustic startle (PPI), a type of preattentional sensory processing (Ralph et al., 2001). PPI deficits in DAT -/- mice are reduced by 5

psychostimulant drugs at doses that cause impairments in DAT +/+ mice (Yamashita et al., 2006), but also the selective NET blocker nisoxetine, suggesting that the effects of psychostimulants on PPI might result from actions at NET. Intracerebral injection studies show that the actions of NET blockers in DAT -/- mice are due to effects in the prefrontal cortex (Arime et al., 2012). DAT -/- mice also have impairments in the cliff avoidance reaction (CAR), in which more than 50% of DAT-/- mice fall from an elevated platform while almost no DAT +/+ mice fall. These deficits can either be taken to reflect impulsivity or executive deficits, both symptoms of ADHD. Methylphenidate and nisoxetine also attenuate CAR impairments in DAT -/- mice (Yamashita et al., 2013).

The DAT -/- model thus presents substantial evidence of clear face and predictive validity but there is also some degree of construct validity for the DAT KO model as well. Although large increases in extracellular dopamine were observed in the striatum, there were few changes in dopamine levels in the prefrontal cortex in DAT -/- mice (Shen et al., 2004), disrupting the dopaminergic balance between the prefrontal cortex and striatum. This dopaminergic imbalance appears to have long-term consequences for frontostriatal circuitry by affecting pyramidal cell morphology (Kasahara et al., 2015), associated with reductions in prefrontal BDNF levels (Li et al., 2010). Moreover, selective elevations in prefrontocortical dopamine function by psychostimulant drugs in

DAT -/- mice at doses that do not affect dopamine levels in the striatum improve learning in DAT-/- mice (Takamatsu et al., 2015). The prefrontocortical neurons that underlie these effects have been identified based on patterns of activation by the NET blocker nisoxetine, which activates glutamatergic neurons that project to the nucleus accumbens

(Arime et al., 2012). 6

Based on the foregoing discussion, it is clear that there is substantial evidence that DAT -/- mice constitute a valid model of ADHD. However, the model has yet to be assessed in sophisticated behavioral assays that are more relevant to attentional and cognitive dysfunctions in ADHD. The experiments presented in this thesis address those deficits as part of a broader characterization of this model, providing substantial additional evidence for the face and predictive validity of the model. However, despite the strengths of the model in some respects, there are other shortcomings, particularly in terms of the underlying etiology of ADHD. Although genetic alterations in DAT are observed in ADHD (Asherson and Consortium, 2004), either in association with ADHD itself or ADHD endophenotypes, the effect size is not large, implicating a polygenic and multi-causal basis of ADHD. Moreover, complete deletion of DAT in humans is extremely rare and deleterious (Kurian et al., 2009). The range of variation in heterozygous DAT KO mice (DAT +/- mice) is within the range of variation in DAT expression normally observed in humans (Wilson et al., 1996). However, DAT +/- mice do not exhibit the profound deficits observed in DAT -/- mice, although they do exhibit some alterations in dopamine function (Gainetdinov et al., 1998; Giros et al., 1996), including a small increase in locomotion that can be observed when a very large number of subjects is studied (Hall et al., 2014a). Other genetic and environmental factors certainly contribute in an additive or interactive fashion to produce ADHD-like phenotypes in humans, perhaps differentially for the various symptoms associated with

ADHD. Thus, it might be possible to recapitulate the DAT -/- phenotype by an additional

“second hit” in DAT +/- mice. This possibility is investigated in Chapter 3, that combines social isolation from weaning with the partial reduction of the dopamine transporter in 7

DAT +/- mice. Social isolation at weaning, termed isolation rearing, produces many of the same behavioral phenotypes observed in DAT -/- mice, including hyperdopaminergic function, locomotor hyperactivity and impaired PPI (Hall, 1998; Hall et al., 1998;

Pietropaolo et al., 2008; Wilkinson et al., 1994), although the magnitude of these deficits are smaller than those seen in DAT -/- mice.

With regard to what mechanisms might be explored in a search for alternative medications for ADHD, Hall and colleagues (Hall et al., 2014b) recently found that antagonism of the serotonin 1B (5-HT1B) receptor ameliorates the profound locomotor hyperactivity observed in DAT -/- mice. Moreover, in contrast to some of the previous results with serotonergic compounds (e.g. Gainetdinov et al. (1999)), it does so without completely suppressing locomotor behavior, perhaps because of much more selective actions on the serotonin system. Moreover, the same drug was without effect on locomotion in DAT +/+ mice, potentially indicating a wide therapeutic window. Chapters

4 and 5 further explore the potential of the 5-HT1B antagonist to reduce ADHD-like phenotypes. These chapters further explore ADHD-like phenotypes, focusing on hyperactivity, CAR deficits (reflecting impulsivity or deficits in risk assessment), and PPI

(a model of preattentional sensorimotor gating). Previous findings are extended in many ways in these studies. DAT +/- mice have not been previously characterized for PPI or

CAR deficits. The effect of sex as a factor has also not been previously characterized. In addition to extending the model and evaluating DAT +/- mice a better potential model than DAT -/- mice behaviorally, the ability of known ADHD treatments as well as the effects of the potential novel treatment, 5-HT1B antagonism, were also assessed. Finally, in Chapter 6, the DAT +/- mice were evaluated in a model that directly assessed attention 8

and impulse control in a model of spatiotemporal discrimination, including the ability of known ADHD treatments to ameliorate ADHD-like deficits and assessment of the potential of 5HT1B antagonism to do so as well.

9

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11

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14

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741-749.

16

Chapter 2

Evaluation of the Validity of Animal models of Attention Deficit Hyperactivity

Disorder (ADHD)

a, b a Yasir H. Saber , and F. Scott Hall , a Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH, USA; b Ninevah College of medicine, Ninevah university, Mosul, Iraq

Manuscript in Preparation for submission to Neuroscience and Biobehavioral Reviews.

17

Abstract

Attention Deficit Hyperactivity Disorder (ADHD) is a common disorder in children, but the symptoms also persist into adulthood for many individuals. Although a number of treatments exist for ADHD, including the widely prescribed psychostimulant medications, as well as some non-stimulant alternatives, not all individuals are well- treated by these drugs. The causes of ADHD include both genetic and environmental factors, but since the condition has a very high heritability, most animal models have focused upon genetic models. The focus of these genetic models has changed over time, along with broad changes in genetic and genomic knowledge. Many newer models are based upon the identification of certain genetic variants associated with ADHD in humans, that are then reproduced in some way in animals. One of the problems with these approaches is that the genetic variants upon which they are based often explain only a very small percentage of the variance associated with the likelihood of developing

ADHD. Indeed, many of the most valid models were based upon a priori considerations derived from the role of certain neurotransmitter systems in ADHD, and the behavioral functions affected in ADHD, as well as the modes of actions of ADHD treatments. Many genetic models of ADHD have involved mutations of the dopamine transporter gene.

This review considers many ADHD models, but focuses upon many of the mutant DAT strains as they appear to provide the most valid animal models of ADHD available.

18

1.0 Introduction

The symptoms of ADHD are heterogeneous, reflecting the underlying heterogeneity of its etiology, which includes both genetic and environmental components

(Bidwell et al., 2017; Faraone & Larsson, 2019; Palladino, McNeill, Reif, & Kittel-

Schneider, 2019). The most recent edition of diagnostic statistical manual for mental disorders (Diagnostic and statistical manual of mental disorders : DSM-5, 2013) subdivides ADHD into three main diagnostic sub-categories: (1) inattentive, (2) hyperactive / impulsive, and (3) combined of inattentive and hyperactive / impulsive.

Although this nosology effectively defines most cases of ADHD, it might be better to think of ADHD as potentially affecting several domains of cognitive and behavior functioning, more in line with the nosology put forward by the Research Domain Criteria initiative (RDoC; see discussion in Baroni and Castellanos (2015)). According to this approach there would be several functional domains that are affected in ADHD in an independent manner that include reward-related processing, inhibition, vigilant attention, reaction time variability, timing and emotional liability. These represent different aspects of the classically-described ADHD symptoms, but also include aspects of impaired executive function and decision making that have also been described in some individuals with ADHD (Pineda-Alhucema, Aristizabal, Escudero-Cabarcas, Acosta-Lopez, & Velez,

2018). Indeed, risky or poor decision-making may be one of the poorest-treated symptoms found in patients with ADHD (Dekkers et al., 2018; Todokoro et al., 2018).

Attention deficit/hyperactivity disorder (ADHD) is one of the most prevalent developmental neuropsychological disorders in children. ADHD has been reported to

19

affect between 5.3 and 11% of school-age children (Lecendreux, Konofal, Cortese, &

Faraone, 2015; Polanczyk, Willcutt, Salum, Kieling, & Rohde, 2014; Visser et al., 2014) and 3.4% of the adult population (Fayyad et al., 2007). The reduction of ADHD incidence in adulthood reflects amelioration of certain symptoms with age in many individuals, in particular hyperactivity and impulsivity, although other cognitive symptoms are more persistent. ADHD incidence has continued to increase over the last decade, especially among adults and females (Fairman, Peckham, & Sclar, 2017). This may reflect more accurate diagnosis, particularly in females, as well as a recognition of adult symptom profiles that are different from adolescents. There may also be changing environmental factors leading to an actual increase in ADHD incidence. ADHD has a negative impact on academic and professional achievement, and social relationships, consequently producing a large emotional and financial burden for patients, families, and communities (de Zeeuw, van Beijsterveldt, Ehli, de Geus, & Boomsma, 2017; Fenesy,

Teh, & Lee, 2019; Rietveld & Patel, 2019; Zendarski, Mensah, Hiscock, & Sciberras,

2019; Zendarski, Sciberras, Mensah, & Hiscock, 2017). Additionally, at least one other psychiatric comorbidity exists in 80% of ADHD patients, and especially concerning is a tendency for increased rates of drug use and drug addiction in adolescents with ADHD

(Biederman, Newcorn, & Sprich, 1991; Romo et al., 2018). This may reflect either common underlying causative factors between these conditions, or the effect of some

ADHD symptoms on choices as to whether to engage in drug-taking and other behaviors.

Core symptoms of ADHD include attentional impairment and hyperactivity, but also impulsivity and deficits in executive function that might directly impact on adolescent decision-making. ADHD has been divided into subtypes based on symptom profiles that 20

differentially involve these symptoms: inattentive, hyperactive/impulsive, and combined

(DSM-V; American Psychiatric Association (2013), but not all symptoms are equally treated by current medications. 69% of children with ADHD are prescribed medication, with psychostimulant drugs like methylphenidate and amphetamine (Ritalin and

Adderall, respectively) being the most commonly prescribed drugs (Punja et al., 2016;

Storebo et al., 2015). Methylphenidate acts through blocking dopamine and norepinephrine transporters in the prefrontal cortex and striatum (Romo et al., 2018; R.

C. Spencer, Klein, & Berridge, 2012). There is some debate as to which site of action is most important, but it is likely to be the prefrontal cortex, as will be discussed in more detail below. Amphetamine acts as a monoamine releaser by reversing plasma membrane monoamine transporter function, producing release of norepinephrine and dopamine in terminal regions. The actions of these drugs on norepinephrine and dopamine in some brain regions are likely to be key factors in their effects on ADHD symptomatology, although effects in other regions may be related to side effects and other negative outcomes.

There has been concern for some time that early use of psychostimulant drugs in children with ADHD may produce adverse long-term outcomes, including addiction.

Psychostimulant use has been suggested to increase the potential for the development of drug addiction and raised concern regarding the use of these drugs on the development of dopaminergic systems when given at an early age in children with ADHD (Gerlach,

Grunblatt, & Lange, 2013; Khan et al., 2017). Although some non-psychostimulant medications like atomoxetine, guanfacine and clonidine are available for the treatment of

ADHD, a relatively high percentage (35%) of ADHD patients do not respond well to 21

these medications (Beherec et al., 2014), and side effects may limit the ability to reach effective therapeutic doses. Accordingly, the development of a novel non-stimulant medication with a more favorable side-effect profile is highly desirable. It should be possible to accomplish this goal by finding a druggable alternative pathway to psychostimulants or atomoxetine to treat or alleviate ADHD symptoms. Of course, one of the difficulties in identifying such an alternative pathway is the limited understanding that we currently have of the way in which even the oldest ADHD treatments, psychostimulant drugs, produce their effects (Rubia et al., 2014). Recent work with a new animal model of ADHD, dopamine transporter knockout (DAT KO) mice, has suggested how psychostimulant drugs may actually work to modulate aberrant corticostriatal neurocircuitry in patients with ADHD. Preclinical studies in laboratory animals are indispensable for understanding the pathophysiology of psychiatric disorders, as well as for developing potential therapeutic approaches. Although other animal models of ADHD have been proposed over the years, they all have substantial short-comings (Arime,

Kubo, & Sora, 2011; de la Pena et al., 2018; Fan, Bruno, & Hess, 2012; Wickens,

Hyland, & Tripp, 2011), and very few of them have concentrated upon all of the core symptoms of ADHD, often focusing primarily upon hyperactivity (Carvalho, Vieira

Crespo, Ferreira Bastos, Knight, & Vicente, 2016; Wickens et al., 2011). Although environmental/experiential factors may contribute to the development of ADHD, and preclinical models for these factors have been developed (V. A. Russell, 2011) most work has focused upon genetic models (de la Pena et al., 2018), partly because of the high heritability estimates for ADHD of between 70 and 90% (Larsson, Chang, D'Onofrio, &

Lichtenstein, 2014). Consequently, this review will consider the currently available 22

genetic models of ADHD, highlighting the behavioral methods used for validation of the models, and comparing those to behavioral outcomes in humans with ADHD. Much focus is given to animal models that involve DAT, for reasons that will become evident.

2.0 Animal models using inbred rodent strains

Given that the exact etiology of ADHD is unknown, several studies have approached modeling ADHD by examining inbred rodent strains that show high levels of motor activity.

2.1 The spontaneously hypertensive rat (SHR)

The inbred SHR strain is the most widely studied animal model of ADHD. SHR

(or SHR/NCrl) is an inbred strain derived from the Wistar-Kyoto (WKY; or WKY/NHsd) rat, which is commonly used as a control strain for comparisons because of its close genetic relationship to the SHR strain. This strain was originally studied for hypertension and liability to develop stroke. However, SH rats also exhibit locomotor hyperactivity

(Knardahl & Sagvolden, 1981; Moser, Moser, Wultz, & Sagvolden, 1988), prior to the onset of hypertension, which led to their description as an animal model of ADHD

(Sagvolden, Pettersen, & Larsen, 1993). SH rats have also been suggested to show a variety of deficits in operant tasks, including impulsive impairments (Wultz &

Sagvolden, 1992) and perseverative deficits (Mook & Neuringer, 1994). Deficits during the extinction portion of an operant task appeared to reflect different deficits in male and female SH rats (Berger & Sagvolden, 1998), the deficit in females appearing to reflect attentional impairments, while the deficit in males resulted from an poorer ability accept 23

delayed reinforcement. Indeed, SH rats have deficits in temporal delay discounting

(Adriani, Caprioli, Granstrem, Carli, & Laviola, 2003), although it must be noted that these animals were tested in adolescence, and that it appeared to be only a subgroup of

SH rats that was impulsive. Despite deficits in many operant circumstances, SH rats do not show deficits in DRL (differential reinforcement of low rates) responding (Bull,

Reavill, Hagan, Overend, & Jones, 2000); indeed, those authors suggested that SH rats do not show ADHD-like deficits in this task and that WKY rats are not good controls for the

SHR strain. Other authors have also questioned the adequacy of these strain comparisons

(Alsop, 2007; Sagvolden et al., 2009).

An important criterion for validity of an ADHD model is the ability of currently approved ADHD treatments to reduce ADHD-like deficits in the model. Thus, the deficits in SHR rats should be reduced by drugs that treat ADHD (i.e. predictive validity).

However, amphetamine does not reduce locomotor activity in SHR rats, rather leading to a greater increase in locomotor activity than control rats (Calzavara et al., 2011).

Amphetamine also impaired social interactions in SH rats in that study which is also an undesirable outcome, and although not a primary symptom, individuals with ADHD do have difficulties in social interactions. Prepulse inhibition (PPI) deficits are found in SH rats, but, again, they are not mitigated by amphetamine (R. Levin et al., 2011). On the other hand, learning deficits produced by perseverative errors in SH rats are reduced by amphetamine (Mook & Neuringer, 1994). This may indicate that only some deficits in

SH rats are ADHD-like.

The underlying cause of the behavioral differences in SH rats are not fully known but differences in the expression of dopaminergic receptors in the striatum are observed 24

(Carey et al., 1998). There are presynaptic changes in the regulation of striatal dopamine release in SH rats (V. A. Russell, 2000). However, dopamine transporter expression in

SH rats is not consistently altered (Leo et al., 2003; Watanabe et al., 1997), so changes in striatal dopamine release dynamics likely involve other mechanisms. There are also differences in the organization of nucleus accumbens modules (Papa, Sagvolden,

Sergeant, & Sadile, 1996) and evidence for altered corticostriatal circuit functional connectivity and activity (Papa, Berger, Sagvolden, Sergeant, & Sadile, 1998; Papa et al.,

2002) in SH rats. Altered transcription factor expression (Papa, Sergeant, & Sadile, 1997) may developmentally regulate changes in dopamine receptor expression and changes in corticostriatal circuitry in SH rats (Papa, Sergeant, & Sadile, 1998). Indeed, there are alterations in the expression of Homer/HOMER isoforms in the prefrontal cortex of SH rats, perhaps indicative of changes in prefrontal glutamatergic function (Q. Hong et al.,

2009). There is also evidence for alterations in monoaminergic modulation of the prefrontal cortex of SH rats, including increased noradrenergic activity in the prefrontal cortex but reduced dopaminergic activity of SH rats (V. Russell, Allie, & Wiggins, 2000;

V. A. Russell & Wiggins, 2000). Differences in dopaminergic function in SH rats appear to result from increased terminal autoreceptor inhibition of DA release (V. Russell, de

Villiers, Sagvolden, Lamm, & Taljaard, 1995). Changes in the prefrontal expression of many genes involved in monoaminergic and corticostriatal function are altered in SH rats

(Qiu et al., 2010).

In summary, although SH rats could be said to have a degree of face validity for

ADHD, there are definitely issues with predictive validity for this model, with several of the deficits not being affected by effective ADHD treatments. Altered corticostriatal 25

function is certainly observed in SH rats, which may suggest that this model has some degree of construct validity, but the similarity of these changes to the changes observed in ADHD remain a matter of debate. One might also expect there to be a greater co- morbidity between hypertension and ADHD if this model well-represented the genetic causes of ADHD, but there do not appear to be any direct links between hypertension and

ADHD, although indirect links through obesity may exist (Fuemmeler, Ostbye, Yang,

McClernon, & Kollins, 2011).

2.2 Selectively bred hyperactive mice

Consistent with the idea that ADHD is the extreme end of a continuous population distribution for ADHD phenotypes, selective breeding using a genetically diverse background to develop mice with an ADHD-like hyperactivity phenotype

(Zombeck, Deyoung, Brzezinska, & Rhodes, 2011). As ADHD patients show motor hyperkinesia at home and school, mice were chosen based on their basal motor activity in their home cages with the 10th generation of selectively bred mice being four times as active as control mice (Majdak et al., 2014; Zombeck et al., 2011). In addition to hyperactivity, selectively bred hyperactive mice have low body weight at adulthood and reproductive issues (Majdak et al., 2014). Although learning should not be directly affected in an animal model of ADHD, it would be expected to be indirectly affected by attentional deficits, hyperactivity and impulsivity, as it is in ADHD patients. Selectively- bred ADHD mice demonstrate impairments at learning the Morris water maze, but these deficits were not attenuated by amphetamine, although a chronic low dose treatment with amphetamine mitigated their hyperkinesia (Majdak et al., 2014). It must be noted here 26

that this is not the way that ADHD medications work, which tend to have immediate beneficial effects, rather than effects which develop over a course of chronic treatment.

However, in terms of face validity, the high activity mice also showed high impulsivity in a GO/NoGO task, as shown by a higher false alarm rate (responses to auditory cues where the responding is inappropriate).Chronic treatment with amphetamine did not attenuate this impulsive deficit either (Majdak et al., 2016). It is noteworthy that high activity mice when tested in the Y maze did not demonstrate deficits in arm alteration

(Majdak et al., 2014), indicating that these mice did not have short term memory deficits or perseverative tendencies. A transcriptome analysis of the striatum in the high activity line identified changes in laterophilin 3 gene transcripts, a gene which has been associated with ADHD in humans. Laterophilin 3 was downregulated, as well as several genes related to excitatory synapse regulation like cofilin and fibronectin leucine rich transmembrane protein 3 (Sorokina et al., 2018). Collectively, high activity mice demonstrate face validity in terms of hyperactivity and impaired impulse control.

Predictive validity was seen in terms of the paradoxical amelioration of hyperkinesia, although the manner of treatment was not similar to ADHD and impulsivity was not affected. Finally, construct validity did show some limited similarities to ADHD based on striatal transcriptome analyses.

3.0 Genetically-modified rodent models

3.1 Genetic modifications targeting the dopamine transporter

Genetic studies have suggested that variation in the dopamine transporter gene might contribute to the high genetic heritability of ADHD (Genro et al., 2008; Grunblatt, 27

Werling, Roth, Romanos, & Walitza, 2019; J. H. Hong, Hwang, Lim, Kwon, & Jin, 2018;

B. Yang et al., 2007). Such relationships have been difficult to detect due to underlying genetic heterogeneity of the disorder and small individual contributions of each genetic difference (Faraone & Larsson, 2019), as well as the likelihood of gene-environment interactions contributing to these effects (Palladino et al., 2019). The DAT gene is not identified in GWAS studies (Demontis et al., 2019), probably for those reasons. DAT missense mutations are associated with ADHD, but also early onset Parkinsonism

(Hansen et al., 2014), and individual mutations in DAT are associated with ADHD,

Autism Spectrum Disorder, Bipolar disorder and Dopamine Transporter Deficiency

Syndrome (DTDS) (Mergy, Gowrishankar, Davis, et al., 2014). Some imaging studies in humans have reported that striatal DAT binding or availability is reduced (Hesse,

Ballaschke, Barthel, & Sabri, 2009), but DAT function has more often has been reported to be increased (Cheon et al., 2003; K. H. Krause, Dresel, Krause, la Fougere, &

Ackenheil, 2003; T. J. Spencer et al., 2007). One study did report reduced midbrain DAT binding (Jucaite, Fernell, Halldin, Forssberg, & Farde, 2005). Higher DAT binding is not always observed in the striatum of patients with ADHD but higher DAT levels may be predictive of a positive therapeutic response to methylphenidate, which lowers DAT expression (la Fougere et al., 2006), as does nicotine (J. Krause, 2008). While this data does not associate reductions in DAT with ADHD, it does clearly indicate that perturbations in dopamine, and DAT, function are associated with ADHD.

Genetic overexpression of DAT leads to loss of dopamine neurons, oxidative stress, and L-dopa-reversible motor deficits (Masoud et al., 2015), rather than ADHD- like deficits. The comparison of this to models with greatly reduced DAT function may 28

indicate that impaired function may be associated with both elevations and reductions in

DAT levels. In any case, a number of genetic mouse models have shown that reduction in

DAT expression or function leads to ADHD-like deficits.

3.1.1 Dopamine transporter knock out (DAT KO) strains

The dopamine transporter is the protein responsible for terminating dopamine signaling by reuptake of dopamine back into the presynaptic terminal. Giros et al. (1996) reported production of the first genetically modified mouse strain in which the DAT

(Slc6a3) gene had been deleted by homologous recombination, finding that homozygous

DAT mutant (knockout) mice were spontaneously hyperactive (Giros, Jaber, Jones,

Wightman, & Caron, 1996). This finding was confirmed in a second line of DAT KO mice (Sora et al., 2001; Sora et al., 1998). Hyperactivity was observed in homozygous

DAT KO mice, but not heterozygous DAT KO mice in these first studies. The rest of the discussion in this section will refer to homozygous DAT KO mice. DAT +/- mice will be considered in a separate section. Unlike wildtype (WT; DAT +/+) control mice, DAT KO mice did not show increased in locomotor activity in response to psychostimulant drugs, including amphetamine. This locomotor hyperactivity was quite profound; the level of activity in DAT KO mice was three times the locomotor activity observed in DAT +/+ mice. The nature of this hyperactivity was also highly repetitive, invariant, and perseverative (Fox, Panessiti, Hall, Uhl, & Murphy, 2013; Ralph, Paulus, Fumagalli,

Caron, & Geyer, 2001). In addition to hyperactivity, subsequent studies demonstrated a variety of behavioral deficits several deficits in DAT KO mice that seemed to reflect

29

various other phenotypes that were also similar to symptoms observed in patients with

ADHD.

Deficits in prepulse inhibition of acoustic startle (PPI) have been consistently observed in DAT KO mice (Arime, Kasahara, Hall, Uhl, & Sora, 2012; Ralph et al.,

2001; Wong et al., 2012; Wong, Sze, Chang, Lee, & Zhang, 2015; Yamashita et al.,

2006; Yamashita et al., 2013). PPI is a preattentional sensorimotor gating phenomenon, and these effects have been interpreted to reflect potential attentional deficits in DAT KO mice, although this is not really a test of attention per se. DAT KO mice have also been shown to have impairments in the cliff avoidance reaction (Yamashita et al., 2013). This task involves placing mice on a small, elevated platform. Normally mice are very cautious about approaching the edges of the platform (i.e. cliff avoidance), and very rarely fall. Thus, the behavior of DAT KO mice is very distinctive in this test. DAT KO mice are very incautious, approaching the edge frequently and reaching over the edge of the platform, resulting in falls, which rarely occur in WT mice. These effects could be interpreted in one of two ways, but both types of impairment are likely relevant to

ADHD. First, this could be interpreted as impulsivity, as a failure to assess the situation adequately, and acting rashly. However, since the falls often occur after a period of considered exploration this probably does not really reflect an impulsive deficit, at least not alone. Secondly, the deficit could reflect a failure to adequately assess the risk of falling, or to incorporate this in to behavioral choices, reflecting some type of deficit in executive function. This interpretation is especially interesting since this type of deficit is seen in ADHD and is often one of the most worrying symptoms to parents of children with ADHD – that they will injure themselves by doing things that are intrinsically risky. 30

If DAT deletion does induce ADHD-like impairments, it would be expected that they would show deficits in learning tasks, although it is likely that such deficits would be non-specific, that is, not due to deficits in learning or memory mechanisms per se.

This has, in fact, been repeatedly observed, beginning with the first description of DAT

KO ADHD-like deficits (Gainetdinov et al., 1999). In that study, DAT KO mice were shown to have deficits in learning in the radial arm maze, which was confirmed in another study (Dzirasa et al., 2009). DAT KO mice also have impairments in novel object recognition (Wong et al., 2012; Wong et al., 2015) the social transmission of food preference test (Wong et al., 2012; Wong et al., 2015), spontaneous alternation (Li,

Arime, Hall, Uhl, & Sora, 2010), an alternation/reversal task (Del'Guidice et al., 2014), and the Morris Water Maze (MWM) (Morice et al., 2007). Deficits in the MWM were interesting, as only slight impairments were seen in a standard version or a reversal, but substantial impairments were seen in a cued version of the task. That the DAT KO mice were impaired at a simpler version of this task is highly illuminating – suggesting that it was some environmentally-influenced aspect of response selection or behavioral control that is disrupted in these mice. This suggests that despite modest impairments in hippocampal LTP and LTD, the deficits are primarily not the result of alterations in learning or memory, but rather incidental to their abnormal behavioral selection tendencies. Indeed, in many circumstances DAT KO mice tend to persist in initial response tendencies. For instance, in the marble burying task DAT KO mice are substantially impaired (burying fewer marbles) (Fox et al., 2013), which would normally be considered a sign of reduced OCD-like anxiety. However, they essentially ignore the marbles, persisting for the entire duration of the task at exploring the cage, which is their 31

initial behavioral response. In tests of depression, including the forced swim test and the tail suspension test, DAT KO mice persist for almost the entire duration of the test at escape attempts (Perona et al., 2008), which is again their initial response, but persists for much longer than it does in WT mice. In the MWM, the normal initial response is to explore the edges of the maze, attempting to escape. DAT KO mice simply persist at this initial strategy (Hall, unpublished observations).

The above data clearly demonstrate that DAT KO mice show symptoms that may represent all of the phenotypes observed in ADHD: hyperactivity, impulsivity, impaired attention, and impaired executive function/decision making. However, many of the tests discussed above do not directly or specifically address these phenotypes, such as impairments in PPI, so that it will be important to assess DAT KO mice in more complex tests of cognitive and attentional function. Nonetheless, DAT KO mice appear to present a much wider range of phenotypes relevant to ADHD than previously-proposed models.

Moreover, many of these deficits can be ameliorated by drugs that are effective in

ADHD, establishing a high degree of predictive validity for this model.

The first demonstrations that locomotor hyperactivity in DAT KO mice could be reversed by drugs that are effective in the treatment of ADHD was shown by Gainetdinov et al. (1999). This included both amphetamine and methylphenidate. Those authors concluded that these effects were mediated by serotonergic effects of these drugs, but this conclusion was likely an error based upon artifactual effects of serotoninergic agents that induce the serotonin syndrome, which are enhanced in DAT KO mice (Fox et al., 2013).

Initial studies with stimulants had not found reductions in locomotor behavior in DAT

KO mice after stimulant administration. However, these studies all habituated the mice 32

for extended periods of time prior to drug administration. Hall, Sora, Hen, and Uhl (2014) showed that cocaine produced locomotor decreasing effects in DAT KO mice after a short period of habituation, but had no effect when mice were fully habituated. Moreover, the serotonin 1B antagonist SB 224289, or heterozygous deletion of the serotonin 1B receptor, reduced locomotor activity in DAT KO mice. This likely reflects actions on some portion of the circuitry outlined in Figure 1, which was developed to explain the actions of other pharmacological treatments on the behavior of DAT KO mice, as discussed below.

The PPI deficits in DAT KO mice are reversed by systemic injections of methylphenidate, which produces impairments in wildtype mice (Yamashita et al., 2006).

Moreover, these effects of non-selective agents appear to be mediated by actions on the norepinephrine transporter (NET) since the selective NET inhibitor nisoxetine also reverses PPI impairments in DAT KO mice (Yamashita et al., 2006). Furthermore, these effects are mediated by actions on NET in the prefrontal cortex, as demonstrated by the ability of microinjections of nisoxetine in the medial prefrontal cortex, but not the nucleus accumbens, to also reverse these deficits (Arime et al., 2012). DAT-induced deficits in the cliff avoidance reaction are also ameliorated by both methylphenidate and nisoxetine (Yamashita et al., 2013). Atomoxetine also reverses some cognitive deficits in

DAT -/- mice (Del'Guidice et al., 2014). These findings fit with the hypothesis that noradrenergic dysfunctions underlie ADHD and are associated with frontostriatal impairments (Biederman & Spencer, 1999).

In addition to the various drugs prescribed for the treatment of ADHD, one might also include nicotine. The high rate of smoking among ADHD patients (van Amsterdam, 33

van der Velde, Schulte, & van den Brink, 2018) indicates that nicotine may be used as self-medication for attentional and cognitive deficits by ADHD patients. Nicotine has been shown to reduce both hyperactivity in DAT KO mice, and to reduce deficits in PPI

(Uchiumi et al., 2013), similar to the effects of other approved ADHD medications in

DAT KO mice described above. These effects were all based on acute nicotine administration. Another study has shown that nicotinic α7 receptor expression in the prefrontal cortex of DAT KO mice is increased by chronic oral nicotine administration, which improves performance in a spatial memory task and mitigates motor hyperactivity

(Weiss, Nosten-Bertrand, McIntosh, Giros, & Martres, 2007; Weiss, Tzavara, et al.,

2007). The Uchiumi et al. (2013) study additionally showed that the effects of nicotine were dependent on nicotinic α7-containing receptors and 5-HT1A receptors. Based on these findings, and to incorporate previous findings with nisoxetine, they proposed a circuit that might underlie these effects. A modified version of this figure is presented in

Figure 1, with addition of the location of 5-HT1B receptors in this circuit that might underlie the effects observed by Hall, Sora, et al. (2014). As can be seen in the figure,

5HT1B receptors are located in many locations that might alter the proposed circuitry, and in particular glutamatergic outputs of the prefrontal cortex. 5HT1B receptors are well known to influence serotonin neurotransmission through actions on axons and axon terminals (Sari, 2004). However, Beliveau et al (2016) found association between neocortical 5HT1B receptor mRNA and 5HT1B receptor density that was not associated with subcortical regions suggesting that some neocortical 5HT1B receptor expression is present on the cell body or dendrites of cortical neurons (Beliveau et al., 2016) .

Consistent with this suggestion, an ultrastructural study by Peddie, Davies, Colyer, 34

Stewart, & Rodriguez, (2008) examined the cellular localization of 5HT1B receptors in the hippocampal dentate gyrus, finding that 18% of 5HT1B immunoreactivity was pre-

Figure 1.

Figure 1: Schematic illustration of the neural circuitry that may underlie the effects of nisoxetine, methylphenidate, and nicotine in DAT KO mice involving the medial prefrontal cortex (mPFC). Stimulants and methylphenidate act to increase extracellular levels of dopamine and norepinephrine in the prefrontal cortex by inhibiting NET. Nicotine acts at α7 nicotinic receptors to induce 5-HT1A receptor-dependent serotonin release. Other serotonin receptors either here, or in other parts of this circuit may influence its function. This includes 5-HT1B receptors in several portions of this circuit. Abbreviations: DAT (dopamine), GluR (glutamate receptors), LC (locus coeruleus), NE (norepinephrine), Pyr (prefrontal pyramidal neurons), VTA (ventral tegmental area), and 5-HT (serotonin). (Figure modeled after Uchiumi, et al. 2013) synaptic (associated within axons or axon terminals), while 65 % was post-synaptic

(associated within dendrites or dendritic spines). 5HT1B labelling was rarely associated with GABAergic markers (Peddie, Davies, Colyer, Stewart, & Rodriguez, 2008).

35

Furthermore, a study investigating the delivery mechanism of GPCRs to dendrites found that most of mechanism of delivery was via lateral diffusion, while the 5HT1B receptor was sent to dendrites in secretory vesicles (Liebmann et al., 2012).

Learning deficits in DAT KO mice are likely indirect in nature, resulting from other cognitive consequences of DAT deletion. Regardless of the underlying nature of those effects, many of these impairments can also be ameliorated by drugs that are effective ADHD treatments. There have been repeated suggestions that DAT polymorphisms or differences in DAT function or expression may be involved in ADHD, as discussed above. The nature of this involvement is not certain, and in fact increases in

DAT in the striatum have been most often found in adolescents and adults with ADHD.

Moreover, since complete elimination of DAT function is certainly not observed in

ADHD, this weakens the case for construct validity except in that DAT KO mice certainly show perturbations in dopaminergic function. The question of whether heterozygous DAT KO mice, with 50% of normal DAT expression may better model

ADHD is considered in the next section. Genome-wide association studies (GWAS) have generally failed to confirm a role for DAT polymorphisms in the genetic contributions to

ADHD (Demontis et al., 2019). Reduced DAT protein expression could be produced by environmental factors or interactions with other genetic or environmental factors that are not captured by standard GWAS approaches (for discussion of these issues see (Hall,

2016)); although, as discussed above, this does not seem to be supported by imaging studies. In any case, additional data suggests that many of the deficits in DAT KO mice arise not just from elimination of DAT, but rather from circuit level changes that involve reduced corticostriatal activity and an imbalance between cortical and subcortical 36

dopamine function. This might still approximate some aspect of the underlying state in

ADHD.

The first indication that other mechanistic changes in DAT -/- mice might model biological changes in ADHD was from microdialysis data showing that although basal dopamine levels in the striatum were increased by 10-fold in the striatum of DAT -/- mice, there were no changes in the prefrontal cortex (Shen et al., 2004). Smaller elevations in the prefrontal cortex are observed using quantitative low perfusion rate microdialysis (Xu et al., 2009). On reflection this was not surprising since the levels of

DAT expression in the prefrontal cortex are quite low (Moron, Brockington, Wise,

Rocha, & Hope, 2002). Structural abnormalities in DAT -/- mice were further investigated using magnetic resonance imaging (MRI) analysis at microscopic resolution, finding that anterior striatum size is smaller in comparison with DAT +/+ mice and contains fewer neurons (Cyr, Caron, Johnson, & Laakso, 2005). There is a specific loss of GABA neurons (Cyr et al., 2003), and reduced medium spiny neuron spine density

(Berlanga et al., 2011) resulting in sporadic dyskinesia in some mice. Changes in GABA function are offset by reductions in presynaptic dopamine function that make most mice asymptomatic. Moreover, imaging approaches have also shown that there is reduced activity (or connectivity) in the projection from the medial prefrontal cortex to the nucleus accumbens (X. Zhang et al., 2010) and reduced coordination between cortical and striatal neuronal ensembles (Costa et al., 2006).

This overall picture accords with studies examining the mechanisms of nisoxetine-induced improvements in aberrant behavioral phenotypes in DAT -/- mice

(Arime et al., 2012). That study firstly showed that nisoxetine activated cells in a number 37

of structures that influence ventral striatal activity, including the subiculum, the nucleus accumbens shell, and the medial prefrontal cortex, as well as the ventral striatum itself.

Structures associated with other striatal circuits were not activated by nisoxetine.

Secondly, the cells that were activated by nisoxetine in the prefrontal cortex were glutamatergic neurons projecting to the nucleus accumbens (but not the ventral tegmental area). That study also noted that basal cFos activity was reduced in the medial prefrontal cortex of DAT -/- mice. This accords with several other findings showing that levels of brain-derived neurotrophic factor were reduced in the prefrontal cortex of DAT -/- mice, but not other brain regions (Li et al., 2010), reductions in the numbers of dendritic spines and dendritic arborization of pyramidal neurons in the medial prefrontal cortex of DAT -

/- mice (Kasahara, Arime, Hall, Uhl, & Sora, 2015), and reduced dopamine mediated neuroplasticity (Xu et al., 2009). These alterations were suggested to result from increased stimulation of prefrontal dopamine D2 receptors, relative to D1 receptors, in

DAT -/- mice. Impaired LTP in DAT -/- mice as restored by amphetamine or nisoxetine, and these effects were dependent on activation of noradrenergic receptors (Xu, Ma,

Spealman, & Yao, 2010). Similar effects on LTP are seen in the hippocampus of DAT -/- mice (Morice et al., 2007). These effects are certainly consistent with the amelioration of various DAT KO-induce deficits by specific and non-specific NET blockers. Moreover, amelioration of PPI deficits in DAT -/- mice were produced by injections of nisoxetine directly into the medial prefrontal cortex, but not the nucleus accumbens (Arime et al.,

2012). If the DAT -/- mouse does in fact model aspects of the underlying constructs of

ADHD (if perhaps not the way that this state is usually produced in patients), then this

38

would imply that ADHD medications primarily act in the prefrontal cortex to normalize corticostriatal function.

Recently, a DAT -/- rat strain has been developed which has high spontaneous locomotor activity that, deficits in prepulse inhibition, impairments in the marble burying test, impaired spontaneous alternation (Adinolfi et al., 2018; Adinolfi et al., 2019; Leo et al., 2018). As noted above, DAT -/- mice have also been found to show all of these same deficits. Locomotor hyperactivity in DAT -/- rats is also attenuated by amphetamine and methylphenidate. The effects of DAT KO on tissue and extracellular dopamine in DAT -

/- rats were also similar to DAT -/- mice. DAT -/- rats also have disrupted BDNF expression (Leo et al., 2018), including reductions in prefrontal BDNF levels like those seen in DAT -/- mice, discussed above. Adinolfi et al. (2018) examined locomotor activity in DAT -/- rats at preadolescent, adolescent and adult ages, finding hyperactivity at both pre-adolescent and adult, but not adolescent, ages (Adinolfi et al., 2018). These important developmental issues have not been very well explored in any of the genetically modified DAT strains of mice. Some behavior in DAT -/- rats differs from that reported in DAT -/- mice. DAT -/- rats have reduced anxiety in the light dark test

(Adinolfi et al., 2019), but bury more marbles than WT rats (Adinolfi et al., 2019). DAT -

/- rats also showed reduced contextual and cue-induced fear conditioning, and more consistent exploration of the conditioned chamber. This may have simply resulted from the subjects’ inability to maintain stillness, which may still reflect ADHD-like traits.

The changes in behavior in DAT -/- mice have at times been described as resembling schizophrenia (Ralph et al., 2001; Wong et al., 2012; Wong et al., 2015), and in those studies dopamine antagonists were effective in reducing some DAT KO-induced 39

behavioral deficits. This is not terribly surprising since some of those are certainly the result of elevated dopamine function. Although in the foregoing discussion the DAT -/- model was discussed as a model of ADHD, it might be better to consider it to be a model of frontostriatal dysfunctions that may overlap several psychiatric diagnoses, including

ADHD (for a more detailed discussion of this idea, see Young, Winstanley, Brady, and

Hall (2017)). However, an argument against this is the normalization of behavior by agents that treat ADHD, that do not have those effects in other disorders.

3.1.2 Heterozygous DAT KO mice (DAT +/- mice)

As discussed in the previous section, one of weaknesses of the DAT -/- model is that the ADHD-like deficits in the model are primarily observed in mice that have no

DAT expression at all, homozygous DAT -/- mice. This degree of impairment of DAT is not observed in people with ADHD, who show much milder perturbations of DAT expression. It might be thought that DAT +/- mice might provide a better model of

ADHD for this reason. DAT +/- mice have one functional copy of DAT gene (Slc6a3) which leads to decreased DAT protein expression of about 50%, slightly reduced tissue dopamine content, reduced D1 and D2 dopaminergic receptor expression, and about two times higher extracellular dopamine levels compared with WT mice (Giros et al., 1996;

S. R. Jones et al., 1998). All of these changes are quite modest in comparison to the changes observed in homozygous DAT -/- mice. DAT +/- mice have been less extensively studied that homozygous DAT -/- mice, in part because initial studies failed to identify behavioral deficits in these mice. Locomotor activity is marginally higher in

DAT +/- mice compared to DAT +/+ mice and was not significantly different in many of 40

the first studies of DAT -/- mice (Giros et al., 1996; Sora et al., 1998), but can be shown to be slightly elevated when a high number of subjects are examined (Hall, Itokawa, et al., 2014). Another study measured activity daily over 4 days, and while WT mice showed between-session habituation, DAT +/- mice did not, so that activity was greater in DAT +/- mice by the fourth day (Spielewoy et al., 2000). Another study found age- dependent differences, whereby DAT +/- mice showed less decline in locomotor activity with age (Dluzen, Ji, & McDermott, 2010), although this was not observed until late adulthood. A more recent study found slightly increased locomotor activity in both adolescent DAT +/- mice and adult DAT +/- mice (Mereu et al., 2017). It is not clear why, but this study found far more consistent deficits in DAT +/- mice than previous studies, as discussed in more detail below.

The majority of studies discussed in the previous section examined homozygous

DAT -/- mice but did not study DAT +/- mice. This lack of interest in studying DAT +/- mice came in part from the failure to identify locomotor hyperactivity in DAT +/- mice in initial studies. In additions, some other deficits that are observed homozygous DAT -/- mice are not observed in DAT +/- mice, including impaired PPI (Mereu et al., 2017;

Ralph et al., 2001), and impairments in the marble burying task (Fox et al., 2013).

However, some other changes, such as immobility in the tail suspension test, are observed equally in DAT -/- mice and DAT +/- mice (Perona et al., 2008).

One way to interpret the behavioral changes in the two genotypes of DAT mice could be that DAT -/- mice show much more profound deficits, but that DAT +/- mice might show more modest deficits, but these may also be more similar in magnitude to those observed in ADHD. In support of this view, some cognitive deficits have been 41

observed in DAT +/- mice, including impairments in novel object recognition (Franca et al., 2016; Mereu et al., 2017), deficits in the reversal stage of an attentional set-shifting task (Cybulska-Klosowicz, Dabrowska, Niedzielec, Zakrzewska, & Rozycka, 2017;

Cybulska-Klosowicz, Laczkowska, Zakrzewska, & Kaliszewska, 2017), and impairments in measures of attention and impulsivity in the 5-Choice serial reaction time task (5-

CSRTT) (Mereu et al., 2017). In the 5-CSRTT task mice must attend to 5 lights on each trial, one of which will be lit, signaling that response in the corresponding nose-poke hole will result in reinforcement. DAT +/- mice demonstrated impulsivity compared to DAT

+/+ mice, responding more often before the light was illuminated (increased premature responses), and also impaired attention based on reduced response accuracy after the light was illuminated (Mereu et al., 2017).

Like the impairments discussed in the previous section for homozygous DAT -/- mice, some impairments in DAT +/- mice can be ameliorated by drugs that are effective in the treatment of ADHD. This has been less well-explored than in DAT -/- mice, in part due to fewer behavioral changes being identified in DAT +/- mice. Nonetheless, it has been shown that low dose amphetamine treatment for five days (0.375 mg/kg IP) normalized DAT +/- performance in a modified version of the 5-choice serial reaction time task test (Mereu et al., 2017), although the lack of adequate experimental controls in that study make it difficult to separate the effect of amphetamine from improvement simply due to continued improvement in the task with repeated testing. That study also showed reductions in hyperactivity after amphetamine treatment in DAT +/- mice.

The underlying biological change, aside from reduced DAT expression, and other compensatory changes in dopaminergic neurons, that might underlie behavioral changes 42

in DAT +/- mice have not been fully explored either. However, several biochemical changes have been observed in DAT +/- mice that may relate to the sort of changes observed in corticostriatal circuits discussed above for DAT -/- mice. These changes include reduced expression of Homer1a mRNA in the prefrontal cortex (Mereu et al.,

2017), which likely reflects changes in glutamatergic signaling because of the role of

Homer isoforms in prefrontal cortex-mediated cognitive functions. Importantly, deficits in Homer1a expression were also normalized by amphetamine treatment. DAT +/- female mice also have deficits in the induction of early growth response protein 2 expression, which is responsible for induction of synaptic protein expression in the orbitofrontal cortex and dorsomedial striatum (Cybulska-Klosowicz, Dabrowska, et al., 2017).

Some work has also been done in a new transgenic DAT KO rat line in DAT +/- rats. Like DAT +/- mice, DAT +/- rats do not show the profound disruptions of dopamine dynamics that are observed in DAT -/- rats (Leo et al., 2018). They do show some modest changes however, like modestly increased basal extracellular dopamine levels in the striatum. DAT +/- rats also do not show many of the characteristics of DAT -/- rats, like locomotor hyperactivity and impaired PPI. DAT -/- rats are hyperactive, but do not show enhanced preference of a novel chamber (Adinolfi et al., 2018). By contrast DAT +/- rats are not hyperactive but show persistent preferences for a novel chamber (Adinolfi et al.,

2018), while DAT +/+ rats demonstrate a transient preference for the novel chamber.

This persistent activation by novelty might be considered to be an ADHD-like trait. Like

DAT +/- mice, when DAT +/- rats are examined more closely they do show some tendencies toward increased activity compared to WT rats, particularly toward the end of test sessions after the locomotion of WT mice has habituated, although these differences 43

are much smaller than those seen in DAT -/- rats. These behavioral alterations in DAT +/- rats may alter behavior in other circumstances too. In a social preference task DAT +/- rats showed normal attraction to a social stimulus rat but were more passive in their social interactions (Adinolfi et al., 2019). Whether this reflects some type of social anxiety, despite their normal social preference, or something else is uncertain, but hippocampal and hypothalamic norepinephrine levels were increased. Anxiety was reduced in DAT +/- rats in the light dark test, and contextual and cued fear conditioning were unaltered (Adinolfi et al., 2019) so differences in social interaction do not seem to reflect a general increase in anxiety. Certainly, more needs to be done to characterize this strain, but there are some indications that DAT +/- rats, like DAT +/- mice, show some phenotypes characteristic of ADHD, without the more extreme perturbations observed in homozygous rats

3.1.3 Other genetic modifications targeting DAT

The previous sections discussed rodents with gene deletions of DAT that produced elimination of DAT or a 50% reduction in DAT expression. Another informative genetically-modified line is the DAT knock down (DAT KD) strain of mice

(Zhuang et al., 2001). These mice have just 10 % of the normal (e.g. WT) DAT protein expression, which causes a moderate increase in extracellular dopamine of about 70%.

These mice demonstrate high unhabituated locomotor activity and amphetamine causes dose-dependent attenuation of locomotor hyperactivity (Zhuang et al., 2001).

Furthermore, DAT KD mice express impulsivity in several cognitive tests. Much of the work with this strain made explicit comparisons of their behavior to bipolar disorder in 44

humans, including exploratory behavior measured in the behavioral pattern monitor

(Young, van Enkhuizen, Winstanley, & Geyer, 2011). This pattern included increased transitions, increased hole-poking and reduced Spatial d, a measure of the spatial distribution of behavior. This behavioral pattern was similar to bipolar patients observed in a human version of the behavioral pattern monitor. DAT KD mice also made riskier choices in a mouse version of the Iowa Gambling Task (IGT) (Young et al., 2011), and both DAT KD and individuals with bipolar disorder show riskier choices in mouse and human versions of the IGT (van Enkhuizen et al., 2014). Deficits in DAT KD mice were also associated with high reward sensitivity. There is more evidence that differences in striatal DAT expression may underlie effects in bipolar patients than there is evidence such differences are involved in ADHD. Unlike the status of DAT function in ADHD, bipolar patients do show consistent reductions in striatal DAT expression (Anand et al.,

2011), and a genetic polymorphism associated with bipolar disorder reduces DAT cell- surface expression (Horschitz, Hummerich, Lau, Rietschel, & Schloss, 2005). However, although studies of DAT KD mice have focused on comparisons to bipolar disorder, it is important to note that there is a substantial comorbidity between bipolar disorder and

ADHD, although the extent of that comorbidity is difficult to assess because of symptom overlap and differing time-courses of each disease (Udal et al., 2014).

Using an N-ethyl-N-nitrosourea (ENU)-driven mutagenesis screening approach, a point mutation was identified in Exon 3 of the DAT coding sequence that produced T/G mutation at nucleotide position 471 and an amino acid substitution of lysine for asparagine at position 157 (N157K) (Vengeliene et al., 2017). This mutation was studied both in vitro and in vivo in N157K rats. This point mutation interfered with the cell- 45

surface expression of DAT, almost completely eliminating striatal DAT expression and uptake, an overall pattern of effects that is very similar to homozygous DAT KO mice.

Indeed, like DAT KO mice, extracellular dopamine levels were greatly increased in the striatum of N157K rats, but the prefrontal cortex was relatively unaffected. Behaviorally,

N157K rats were hyperactive in a novel environment and during the dark phase of a circadian rhythm test. They also showed impaired freezing in a test of conditioned foot- shock, although this might represent just an inability to remain still (like some other strains with reduced DAT function discussed above). N157K rats had reduced consumption of sucrose (unlike DAT KO mice (Perona et al., 2008) and were impaired in an autoshaping test. While this latter test, along with the sucrose consumption test, might be interpreted to reflect reduced function of positive valence systems, it seems more likely that this was just impaired ability to attend to appropriate stimuli during the test, as has been observed for other DAT mutant strains. Similar explanations likely account for

N157K impairments in social interaction and novel object exploration. Importantly, hyperactivity was reduced by both amphetamine and atomoxetine. Thus, this model, although proceeding from an initially different basis, confirms many of the findings of previous studies in DAT KO and DAT KD mice. In terms of validity as an animal model of ADHD, the N157K rat has a similar degree of face and predictive validity to those previous models, but still lacks substantial construct validity in terms of overall effects on

DAT function, although it further supports the potential involvement of DAT perturbations in ADHD-like deficits.

Another genetically modified mouse strain attempted to proceed from an initial point that may have greater construct validity, the identification of functional DAT 46

variants that might have altered expression in ADHD (for discussion of these variants see

Mergy, Gowrishankar, Davis, et al. (2014)). One of the variants identified a nonsynonymous nucleotide polymorphism that produced an alanine to valine substitution at position 599 (Mazei-Robison, Couch, Shelton, Stein, & Blakely, 2005). This mutation did not alter DAT reuptake kinetics or cell-surface expression but produced an outward

“leak” of cytoplasmic DA, termed anomalous DA efflux (ADE), an effect that was enhanced by depolarization (Mazei-Robison et al., 2008). ADE was associated with

CAMKII mediated hyperphosphorylation of DAT (Bowton et al., 2010), a mechanism that is important for reverse transport triggered by amphetamines (Fog et al., 2006).

Interestingly, ADE can be eliminated by DAT reuptake blockers, and this mutation prevents amphetamine from producing ADE-like dopamine release, (i.e. acting as a dopamine releaser) and instead amphetamine acts as a reuptake blocker, preventing ADE.

A DAT Val599 mouse model was made to explore the potential of this mutation to model behavioral aspects of ADHD (Mergy, Gowrishankar, Davis, et al., 2014). These mice, like other strains with reduced DAT function, had greatly elevated basal extracellular dopamine levels (Mergy, Gowrishankar, Gresch, et al., 2014), but unlike other DAT strains this strain does not exhibit hyperactivity, but does show substantially reduced rearing and unusual darting behavior. Thus, despite the interesting alterations in

DAT function shown in these mice, at first glance, they do not seem to exhibit ADHD- like hyperactivity. Whether they show other ADHD-like behavior remains to be seen.

47

3.2.0 Genetic modifications targeting other genes in rodent models

As discussed above, the evidence from humans for a role of DAT in ADHD is complicated. Firstly, there has not been consistent evidence for an association between the most well-studied candidate polymorphisms and ADHD, nor have GWAS identified

DAT as being associated with ADHD. However, more detailed searches for polymorphisms in DAT have identified numerous mutations that are associated with

ADHD, as well as other frontostriatal disorders that share some similar phenotypes, like bipolar disorder. Most of these mutations are rare, but certainly indicate a potential role for DAT in ADHD, just not one mediated by a common genetic polymorphism. Rodent genetic studies, using several different approaches, have consistently found that modifications of DAT function (mostly impaired DAT function) are associated with

ADHD-like phenotypes that can eb reversed by effective ADHD treatments.

Candidate gene studies and GWAS have identified associations between numerous other gene polymorphisms and ADHD, reflecting both the polygenic nature of the disorder and its underlying heterogeneity, both genetic and phenotypic. Consequently, a number of genetically-modified mouse strains have been created to study the potential role of these genes in ADHD-like phenotypes, and as potential models for the role of those genes in ADHD. These have been discussed in detail in a recent review (de la Pena et al., 2018), which made clear the shortcoming of many models, as well as, perhaps, the overall tendency of the field to focus upon hyperactivity as the primary definition of an

ADHD-like phenotype. Indeed, many of the studies that claimed to show deficits in

“inhibitory” control, really just found deficits in locomotor habituation. A great number of the genetically-modified strains have been identified as models of ADHD based solely 48

upon the observation of locomotor activity, including the P35 KO mouse (Drerup et al.,

2010; Krapacher et al., 2010), the PER1 KO mouse (Huang et al., 2015), the casein kinase 1  (CK1 OE) over-expressing mouse (Zhou et al., 2010), the casein kinase 2 KO mouse (Rebholz, Zhou, Nairn, Greengard, & Flajolet, 2013), G protein–coupled receptor kinase–interacting protein-1 (GIT1) KO mouse (Won et al., 2011), diacylglycerol kinase β (DGKβ) KO mouse (Ishisaka et al., 2012; Kakefuda et al., 2010), type 5 G protein β subunit KO mouse (K. Xie et al., 2012), and the nitric oxide synthase 1 KO

(Gao & Heldt, 2015). Several of these mouse strains have also shown paradoxical reductions in locomotion in response to psychostimulant medications, including the P35

KO mouse (Drerup et al., 2010; Krapacher et al., 2010), the casein kinase 1  (CK1 OE) over-expressing mouse (Zhou et al., 2010), and the GIT1 KO mouse (Won et al., 2011).

Some of these strains have also been shown to have learning deficits, but not necessary in any manner specific to ADHD.

Thus, for these models to really be considered to model ADHD a wider range of

ADHD phenotypes needs to be assessed, and the ability of a range of known ADHD medications to reverse ADHD-like deficits in the models needs to be assessed. The models discussed below have been more extensively examined in both regards.

3.2.1 Neurokinin 1 receptor KO (NK1R KO) mouse

The neurokinin 1 receptor (NK1R, or tachykinin receptor 1; TACR1) is a G- protein coupled receptor which is abundantly found in the CNS in neuronal cell bodies and dendrites and is activated by neurokinins like substance P. A polymorphism of the human TACR1 gene, coding for neurokinin1recepor, is a candidate gene for ADHD 49

(Sharp et al., 2014). Mice with functional deletion of the Tacr1 gene (the NK1R KO mouse) ((De Felipe et al., 1998); see summary in Yan, Hunt, and Stanford (2009)) are hyperactive (Fisher, Stewart, Yan, Hunt, & Stanford, 2007; Herpfer & Lieb, 2005; Porter et al., 2015; Yan et al., 2010), and this hyperactivity can be reversed by amphetamine

(Yan et al., 2010) and atomoxetine (Pillidge, Porter, Vasili, Heal, & Stanford, 2014).

Basal and K+-stimulated norepinephrine release in the prefrontal cortex is increased in

NK1R KO mice (Fisher et al., 2007; Herpfer, Hunt, & Stanford, 2005; Herpfer & Lieb,

2005; Yan et al., 2010). K+-stimulated responses are the same after an initial pulse, but not subsequently, indicating that some type of feedback inhibition is altered in NK1R KO mice. This role for prefrontal norepinephrine in NK1R KO mice is interesting in light of the role of prefrontal NET in the ADHD-like phenotypes observed in DAT KO mice. In addition to these changes in prefrontal noradrenergic function in NK1R KO mice, there are also substantial changes in response to serotonergic agents (Froger et al., 2001), including reduced effects of 5HT1A receptor agonist-induced inhibition of raphe cell firing, and increased cortical serotonin overflow in response to paroxetine.

In the 5-CSRTT NK1R KO mice initially had more premature responses that WT mice, and exhibited more perseverative responses (e.g. repeated nose-pokes after a correct response), but did not otherwise demonstrate impairments in accuracy or other measures (Dudley et al., 2013). These findings were replicated in another study, which further showed that the premature responses depended on an unpredictable intertrial interval (Porter et al., 2015). Effects on premature responses were dependent on some epigenetic factors as well, based upon whether the mothers of homozygous offspring were heterozygous or homozygous, although perseverative responses were apparent in 50

either case. Differences based on maternal genotype were also observed in WT mice. In this study omissions were increased in NK1R KO mice, but only when tested in the morning. Yet another study replicated 5-CSRTT findings in NK1R KO mice showing that they depended in part on predictability of the intertrial interval (Yan et al., 2011), including effects on perseveration and premature responses. Moreover, under the variable intertrial interval schedule used in this study the probability of omissions was also increased in mutant mice, as well as the latency to make correct responses, while accuracy was slightly reduced. In this study, similar effects on omissions, perseveration and the correct latency were also observed at a long intertrial interval. Another study replicated many of these effects, including increased omissions in NK1R KO mice, and demonstrated that many of these effects were reduced by repeated testing (Weir et al.,

2014), which may be why some studies have seen weaker effects of NK1R KO (Pillidge,

Porter, Vasili, et al., 2014).

The increased perseverative responses of NK1R KO mice are reduced by methylphenidate (Pillidge, Porter, Young, & Stanford, 2016; Yan et al., 2011), although not under all conditions, and not all impairments are ameliorated (Yan et al., 2011). This effect occurred at a dose that did not otherwise affect behavior of NK1R KO, or WT mice, in the 5-CSRTT, although at slightly higher doses behavior was impaired in a variety of ways in both genotypes, including the number of omissions, false alarms and hit rates. Similarly, guanfacine was found to reduce the percent omissions by NK1R KO mice at a low dose, but at slightly higher doses increased omissions in both WT and

NK1R KO mice (Pillidge, Porter, Dudley, et al., 2014). Premature responses are

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significantly reduced in NK1R KO mice, but not WT mice, by atomoxetine (Pillidge,

Porter, Vasili, et al., 2014).

NK1R KO mice have also been assessed in the 5-choice continuous performance task (5-CCPT) (Porter, Pillidge, Stanford, & Young, 2016), which adds a signal detection component to the 5-CSRTT. NK1R KO mice took fewer trials to reach criterion during training and completed more daily trials. As for the 5-CSRTT, during early stages of training they also had more premature responses, and in this case also had a lower hit rate and a lower accuracy. They also exhibited more perseverative responses throughout testing. However, after training in the 5-CCPT, when tested in an extended test session,

NK1R KO mice had a lower probability of emitting false alarms and a higher sensitivity index, consistent with a reduced tendency to emit inappropriate “go” responses to “nogo” stimuli. Perseverative responses remained high and the latency to make both correct and incorrect responses was slightly longer. Overall, these effects are somewhat consistent with the previous 5-CSRTT results, and generally show that NK1R KO mice show small, but observable effects in models of attention and impulsivity, but primarily early in learning of these tasks or when conditions are changing.

Of potential interest by comparison to SHR rats, NK1R KO mice are also hypertensive (Moyes, Stanford, Hosford, Hobbs, & Ramage, 2016). These mice also show other similarities to some other strains that were previously discussed, including a smaller body size. Despite this smaller body size these mice have increased body fat, particularly when given a high fat diet. This is interesting given the previously mentioned connections between obesity and ADHD.

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3.2.2 Coloboma (SNAP25 knockout) mouse

SNAP25 is one of the proteins that forms the SNARE protein complex that regulates exocytotic-mediated neurotransmitter release (Z. Xie et al., 2017). In addition to this role of SNAP25 neurotransmission, SNAP25 also has a role in postsynaptic receptor trafficking (Wang & Tang, 2006). SNAP25 affects short-term neural plasticity that that is involved in information processing, working memory, and decision making (Antonucci et al., 2013). Several studies have shown an association between SNAP25 polymorphisms and ADHD (Barr et al., 2000; Bidwell et al., 2017; Brophy, Hawi, Kirley, Fitzgerald, &

Gill, 2002; Faraone & Mick, 2010; Herken et al., 2014; Ye et al., 2016; H. Y. Zhang,

Zhu, Chang, & Chen, 2008). The Coloboma mutant mouse was created by neutron irradiation that caused 2 cM deletion of mouse chromosome 2, disrupting about 20 genes, including Snap25 (Hess, Jinnah, Kozak, & Wilson, 1992; Heyser, Wilson, & Gold,

1995). The original mutant was characterized for developmental eye abnormalities

(Theiler & Varnum, 1981), and was subsequently shown to produce a variety of developmental abnormalities. Indeed, the homozygous mutation is lethal, prompting the study of heterozygous Coloboma mutant (Cm/+) mice, which were found to be spontaneously hyperactive (Hess et al., 1992; Heyser et al., 1995). SNAP25 expression is reduced by 50% in these mice (Hess et al., 1992). The locomotor hyperactivity in Cm/+ mice was genetically rescued by replacing the mutant Snap25 gene with a wild-type gene

(Hess, 1996), demonstrating that it was the Snap25 mutation that produced these effects and not the other genes altered in Cm/+ mutants. A broader behavioral characterization of these mice found that in addition to hyperactivity, Cm/+ mutants had impaired PPI and impaired learning of an odor conditioning task, but also impaired motor coordination and

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balance (Gunn, Keenan, & Brown, 2011). Despite these broad abnormalities in Cm/+ mice, they were also found to have impaired attention and impulsivity (Bruno et al.,

2007), as determined by latent inhibition and delayed reinforcement deficits. The interpretation impulsivity in the delayed reinforcement task is complicated by the indifference of Cm/+ to the aversive taste of quinine, which was used in the task, so this may reflect a sensory deficit, rather than a cognitive deficit. Cm/+ mice also have a deficit in novel object recognition which should not be affected by the same type of sensory deficit (Braida, Ponzoni, Matteoli, & Sala, 2016), but given the wide range of deficits in these mice, it is difficult to say what type of deficit this reflects.

Supporting the idea that the Cm/+ mutant might model some aspects of ADHD, the hyperactivity in these mice was reduced by amphetamine (Hess, Collins, & Wilson,

1996). However, in that same study, methylphenidate increased activity in Cm/+ mice, inconsistent with the effects of methylphenidate on hyperactivity in ADHD. Some of the hippocampal neurophysiological changes produced by the Snap25 mutation in Cm/+ mice could be genetically rescued by re-expression of the Snap25 wild-type gene or restored by amphetamine treatment (Steffensen, Henriksen, & Wilson, 1999). The effects of amphetamine on locomotion in Cm/+ mice are dopamine D2 receptor dependent (Fan

& Hess, 2007; Fan, Xu, & Hess, 2010). This is substantially different from the proposed mechanism of psychostimulant effects in DAT KO mice.

Although these mice show a much broader range of physiological and neurochemical disruptions than other models, they do show changes in systems and circuits thought to be involved in ADHD. Neurotransmission was broadly altered in

Cm/+ mice, albeit in a region-dependent manner, but included alterations in dopamine, 54

serotonin, glutamate and corticotropin releasing factor transmission (Raber et al., 1997).

In contrast to reduced dopamine function, there is evidence for enhanced norepinephrine function in Cm/+ mice (M. D. Jones, Williams, & Hess, 2001a, 2001b). It is perhaps a bit surprising that hyperactivity would be observed in animals with reduced dopamine function, but the hyperactivity in Cm/+ mice was actually associated with elevated noradrenergic function (M. D. Jones & Hess, 2003).

In summary, Cm/+ mice had some phenotypes that have been associated with

ADHD, but many of the results were inconsistent with the condition. This is not too surprising given the wide range of developmental and physiological consequences of the mutation. Given the known roles of SNAP25, it might be that that a more selective

Snap25 mutation, perhaps only affecting its role in certain aspects of neurotransmission in particular neural systems or regions, might make a better model and bear more similarity to the polymorphisms underlying the genetic associations that have been found for this gene with ADHD (Barr et al., 2000; Bidwell et al., 2017; Brophy et al., 2002;

Faraone & Mick, 2010; Herken et al., 2014; Ye et al., 2016; H. Y. Zhang et al., 2008).

3.2.3 Mutant Thyroid Hormone Receptor β Knock-in (TRβPV KI) mouse

Several reports have found evidence for an association between prenatal and postnatal thyroid function disruption and ADHD (Chen et al., 2018; Drover et al., 2019;

Fetene, Betts, & Alati, 2018; Kuppili, Pattanayak, Sagar, Mehta, & Vivekanandhan,

2017). Many different mutations in the Thyroid Hormone Receptor β gene (THRB) are associated with a genetically-mediated resistance to thyroid hormone (RTH) condition that has a high rate of comorbid ADHD diagnosis (Kopp, Kitajima, & Jameson, 1996). 55

Point mutations associated with this condition (Meier et al., 1992; Parrilla, Mixson,

McPherson, McClaskey, & Weintraub, 1991), have been used to generate TRβPV KI strains (Kaneshige et al., 2000; McDonald et al., 1998) that show thyroid abnormalities characteristic of RTH and consequent physiological effects. In addition to research investigated thyroid function, phenotypes relevant to ADHD have also been examined in several studies.

Hyperactivity was observed in the first TRβPV KI strain (McDonald et al., 1998), but only in male mice. There were no differences in basic motor function (rotarod and screen hang tests). TRβPV KI mice were slower to learn operant responding in an autoshaping task, but after acquiring criterion showed no differences in a simple reaction time task or a go/no-go task. The data for the operant tasks was not analyzed separately for males and females. Since females did not show locomotor differences, it is possible that this obscured effects on other phenotypes that were sex-dependent, perhaps only occurring in males. Additionally, for operant tasks animals were food deprived.

Importantly, after food deprivation male TRβPV KI mice were no longer hyperactive, and instead were hypoactive compared to control mice. No difference was again observed in females. The authors emphasized that the observation of ADHD-like phenotypes

(hyperactivity) only in males was similar to ADHD. However, as has been argued, this view may misrepresent the actual nature of sex-differences in ADHD, which may be more a matter of qualitative differences in symptom presentation between sexes.

In another TRβPV KI strain (Kaneshige et al., 2000), male TRβPV KI mice had increased activity, but not initially; hyperactivity was only seen after several test sessions, indicative of reduced within-session and between-session habituation (Siesser, Cheng, & 56

McDonald, 2005). By contrast, locomotor activity was decreased in female TRβPV KI mice due to greater within-session and between-session habituation. TRβPV KI mice took longer to learn a vigilance task of sustained attention but also appeared to have enhanced motivation (as determined by the rate of response on an FR1 schedule during training). After reaching criterion, mice were challenged with shorter cue durations, which degraded performance. Methylphenidate did not improve this performance in any group, although male TRβPV KI +/+ mice were resistant to the effects of methylphenidate.

Another study used a TRβPV KI strain in which the effects were limited to the pituitary (Zhu et al., 1999). These mice had similar habituation-dependent hyperlocomotion as Siesser et al. (2005), but also found that TRβPV KI mice were more active after saline injections, and methylphenidate reduced this hyperactivity (Siesser,

Zhao, Miller, Cheng, & McDonald, 2006). In a simple sustained attention task TRβPV KI

+/+ mice performed fine when the reinforcer was large but performed more poorly when the reinforcer size was reduced. This may reflect altered motivation, and perhaps greater negative contrast, since they had higher progressive ratio break points, but only for a large reinforcer. They were also more persistent during extinction, when the reinforcer was replaced with water. This was interpreted as a measure of impulse control, although other explanations are quite likely. However, they were also shown to have impairments in delay discounting (more choices for the smaller reward as the delay increased). In summary, these mice appear to have alterations in motivation, decision-making and impulsivity that are similar to ADHD.

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These studies are interesting for several reasons. Firstly, they are based upon human mutations that are associated with ADHD. However, the causal factors underlying

ADHD development are quite different from the ADHD population at large and associated with a broad range of physiological effects associated with the thyroid dysfunction. Moreover, the two different constructs used to produce KI strains produced quite different effects on locomotor hyperactivity. Given the similarity of the findings in the second strain to those observed after selective activation of the transgene in the pituitary, it seems likely that different human variants may have tissue selective effects.

This would accord with the large variation in symptom severity found with different

THRB mutations. Although these strains provide a good model for how human variation may be translated into animal models, including perhaps the large variation in outcomes from similar modifications of the same gene, they do not provide evidence that THRB mutations effectively model ADHD - locomotor hyperactivity was observed under some limited conditions in these mice, but most other tests did not find ADHD-like phenotypes.

3.2.4 Adhesion G-protein coupled receptor L3 KO (ADGRL3 KO) mice

Latrotoxin is a component of black widow spider’s venom that causes rapid massive release of several neurotransmitters via actions on ADGRL3 (also known as latrophilin 3; LPHN3) (Sudhof, 2001). Latrophilins, including ADGRL3, are g-protein coupled receptors that also have cell-adhesion properties. The effects of ADGRL3 on glutamate, GABA, and acetylcholine release are calcium-independent, while its effects on catecholamine release are calcium-dependent. Genetic polymorphisms of ADGRL3 58

have been associated with ADHD (Arcos-Burgos et al., 2010; Martinez et al., 2016), and may predict patient responses to methylphenidate (Bruxel et al., 2015). ADGRL3 KO mice are hyperactive (Mortimer et al., 2019; Wallis et al., 2012), but cocaine potentiates this hyperactivity (Wallis et al., 2012). ADGRL3 KO mice have impairments in the object recognition task, showing no preference for the novel object, as well as impaired spatial learning in the Barnes maze (Mortimer et al., 2019). In the continuous performance test, ADGRL3 KO mice had a higher false alarm rate during acquisition, and subsequently during probe tests at shorter stimulus durations and when stimulus contrast was reduced. This increased false alarm rate was taken to indicate that ADGRL3

KO mice were more impulsive. ADGRL3 KO mice were also found to be more motivated for food rewards, particularly under more demanding schedules (Orsini et al.,

2016).

These behavioral differences in ADGRL3 KO mice were associated with upregulation of a number of monoaminergic genes in whole brain samples, including

DAT, SERT, the dopamine D4 receptor, the serotonin 5HT2A receptor, and tyrosine hydroxylase, as well as elevations in striatal levels of dopamine and serotonin (Wallis et al., 2012). Greater neurite outgrowth was found in cultured hippocampal and cortical neurons from ADGRL3 KO mice (Orsini et al., 2016). An extensive differential gene expression analysis (Mortimer et al., 2019) found the greatest number of differentially expressed genes in the prefrontal cortex. The striatum and hippocampus were also examined. Among the genes downregulated in the prefrontal cortex were DAT and tyrosine hydroxylase, as well as several cholinergic receptor subunit genes. The reduced expression of DAT and TH in the prefrontal cortex is interesting given the numerous 59

findings in transgenic mouse strains that link DAT and the prefrontal cortex to ADHD- like phenotypes, including circuitry that may involve prefrontal acetylcholine function.

3.2.5 P35 (cyclin-dependent kinase 5 cofactor p35) KO mice

CDK5 is a proline-directed serine/threonine kinase that is predominately active in postmitotic neurons and, among its known functions, regulates dopamine release, synthesis and downstream signaling (Hisanaga & Saito, 2003; Jeong et al., 2013;

Kesavapany et al., 2004; Mlewski, Krapacher, Ferreras, & Paglini, 2008). It is noteworthy that interfering with CDK5 function by pharmacological inhibition, brain- wide conditional inhibition of the Cdk5 gene or viral mediated Cdk5 knockdown targeting the dorsolateral striatum, all impair D1-dependent long-term potentiation in the dorsolateral striatum (Hernandez et al., 2016). A recent study revealed a genetic association of a Cdk5 polymorphism with ADHD (Maitra, Chatterjee, Sinha, &

Mukhopadhyay, 2017). The deletion of the Cdk5 gene causes massive embryonic abnormalities and is lethal, so to manipulate Cdk5 function without interfering with it is expression Chae et al. (1997) targeted the CDK5 cofactor P35. P35 is a synaptosomal protein involved in exocytotic release of neurotransmitters, including dopamine. While

CDK5 KO is lethal, P35 KO is not (Chae et al., 1997). P35 KO mice have alterations in the laminar structure of the cortex through effects on neuronal migration and abnormalities in neuronal circuits underlying motor and reward function, as assessed with positron emission tomography (Chae et al., 1997), as well as broader anatomical abnormalities of the corpus callosum (Drerup et al., 2010; Kwon, Tsai, & Crandall,

1999). Alterations in medial prefrontal cortex-nucleus accumbens connectivity and

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monoamine (dopamine and serotonin) function are also observed in P35 KO mice

(Drerup et al., 2010).

Some of these anatomical and neurochemical changes, but not others, are reminiscent of changes thought to occur in ADHD. These mice have not been extensively examined for ADHD-like phenotypes, but P35 KO mice are hyperactive, and this hyperactivity is attenuated by methylphenidate (Drerup et al., 2010; Krapacher et al.,

2010). Additionally, Krapacher et al. (2010) found that locomotor activity was observed in juvenile (postnatal day 35) P35 KO mice, but not adult mice. They also interpreted increased time spent in the open portions of the elevated plus maze as reduced inhibitory control. This finding is interesting, although not sufficient to demonstrate impaired inhibitory control in P35 KO mice.

The only ADHD symptom that has been observed in P35 KO mice is hyperactivity. The model certainly deserves further investigation of other ADHD-like phenotypes, particularly considering some of the changes in frontostriatal function that have been observed in this strain.

3.2.6 ADF actin depolymerization factor (ADF)/cofilin double mutant mice

Actin is one of the most important cytoskeleton proteins and has crucial role in neurotransmitter vesicle formation and exocytosis, as well as postsynaptic receptor trafficking (Hanley, 2014). Actin function is regulated by LIM kinase 1 through control of the ADF/cofilin family members, including ADF and n-cofilin. While n-cofilin is mainly expressed on postsynaptic dendritic spines, ADF is highly expressed presynaptically, implicating it in vesicle formation and exocytosis. N-cofilin appears to 61

compensate for loss of ADF. Consequently, combined elimination of both ADF and n- cofilin are necessary to impair excitatory pyramidal plasticity (Gorlich et al., 2011). In a mouse mutant combining constitutive ADF KO and a conditional n-cofilin deletion under the control of a CAMKIIα promotor increases in the size of excitatory synapses and increases in neurotransmitter release in both the striatum and the hippocampus are observed (Rust, 2015). Although these changes in synaptic function might impact a wide range of phenotypes, of relevant to the topic of this review, ADF/cofilin double mutant mice are hyperactive and this hyperactivity can be reduced by methylphenidate

(Zimmermann et al., 2015).

To examine working memory, ADF/cofilin double mutant mice were tested in the radial arm maze and the Y-maze (Zimmermann et al., 2015). Double mutant mice had higher rates of errors (arm returns) in both tests. An interesting result was obtained in the elevated plus maze in that report. Intriguingly, 89% of double mutant mice jumped from the elevated plus maze apparatus (0.5 meters above the ground). Even mice that remained on the plus maze platform showed a strong preference for the open arms over the closed arms. The results of this task are usually interpreted in terms of anxiety, but comparison to CAR deficits in DAT KO mice (Yamashita et al., 2013) are difficult to avoid.

Consistent with this type of impairment, which may reflect impulsivity or impaired decision-making, treatment with methylphenidate reduced the number of mice falling and increased the time that the spent on the platform without falling off (Zimmermann et al.,

2015).

The mechanisms underlying these effects are not entirely certain, but changes in striatal or cortical glutamatergic function are likely candidates. Cre expression underling 62

the conditional knockout of cofilin was found in the cortex, striatum, and hippocampus, but at low levels in midbrain dopamine neurons. ADF/cofilin double mutant mice had reduced numbers of excitatory glutamate synapses in the striatum, but these synapses were larger, and the number of docked vesicles was increased (Zimmermann et al.,

2015). NMDA Glutamatergic antagonism with MK801 reduced hyperactivity in

ADF/cofilin double mutant mice, suggesting that there was a functional overactivity of glutamatergic afferents that contributed to these behavioral deficits. This does not necessarily accord with the reduced corticostriatal connectivity observed in other ADHD models, but does certainly suggest that altered glutamatergic projections to the striatum may be involved in ADHD phenotypes. It will be important to explore the glutamatergic function in other brain regions relevant to ADHD, especially the prefrontal cortex.

3.2.7 Guanylyl Cyclase-C 3 KO (GC-C KO) mice

GC-C is a membrane receptor for guanylin and uroguanylin in the gut (Currie et al., 1992). GC-C was recently found to be colocalized with dopaminergic markers in midbrain dopamine neurons (Gong et al., 2011). Furthermore, application of GC-C ligands potentiated excitatory inputs to dopamine neurons. GC-C KO mice were found to be hyperactive, and to have reduced habituation to repeated exposure to an olfactory stimulus. GC-C KO mice were then trained in an operant task where the availability of a water reward was signaled by the presentation of a CS+, while a CS- signaled a shock.

During training on the CS+ GC-C KO mice began licking earlier, and in a manner less linked to the stimulus, often beginning to lick before stimulus onset and stopping only after stimulus offset. Responses to the CS- were also imprecise, with much longer 63

response latencies. Accurate responding in GC-C mice was substantially impaired, much more than control mice, by the introduction of a random delay at the start of each trial.

These patterns indicate that GC-C mice may have impairments of both impulsivity and attention.

A more recent study, controlling for a variety of factors such as litter effects, suggested that they did not find ADHD-like phenotypes in GC-C KO mice (Mann,

Sugimoto, Williams, & Vorhees, 2019). Hyperactivity was not observed in an initial open field test. Subsequently, female GC-C mice showed a slight hyperactivity prior to drug administration, but amphetamine produced a similar locomotor activation in both GC-C

KO and GC-C wildtype mice. A deficit in novel object recognition was observed in these mice, but learning in the Morris Water Maze and conditioned freezing were not affected.

It must be said that although this study claimed to not find any ADHD-like phenotypes in these mice, it did not actually include any approaches that would reveal specific deficits in impulsive or attentional behavior, certainly nothing like what was examined in the previous study.

3.2.8 Phosphoinositide 3 kinase γ KO (PI3Kγ KO) mouse

PI3Kγ is necessary for NMDA-dependent long-term depression in the hippocampus (Kim et al., 2011). Based on these findings PI3Kγ KO mice were investigated in tests of cognitive functions. PI3Kγ KO mice were not hyperactive in this study (Kim et al., 2011), although they were found to be hyperactive in another study

(D'Andrea et al., 2015). In the Kim et al. (2011) study, there were no differences in initial spatial learning in the Morris Water Maze, or in learning a reversal. However, PI3Kγ KO 64

mice spent more time in the previous platform location during a probe trial in which it was removed. This was taken as an indication of reduced behavioral flexibility in PI3Kγ

KO mice. These mice were also slightly impaired in a delayed non-matching to position

T-maze task, a deficit that was also interpreted as resulting from impaired behavioral flexibility. PI3Kγ KO mice also had substantial deficits in the attentional set shifting task

(ASST) for all reversal conditions, the intradimensional shift, and the extradimensional shift (D'Andrea et al., 2015). These impairments were caused by perseverative errors, which could also be considered to result from poor behavioral flexibility. Most importantly, both impaired behavioral flexibility and hyperactivity in PI3Kγ KO mice were reversed by methylphenidate treatment.

The mechanisms underlying these effects have not yet been extensively investigated, but PI3Kγ was extensively colocalized with norepinephrine in the locus coeruleus (D'Andrea et al., 2015). Activation of cAMP response element binding protein

(CREB) in the LC was taken as an indication of increased LC activity, which as associated with increased norepinephrine, but decreased dopamine, in basal forebrain

(frontal cortex and striatum) tissue samples. It is interesting to note that this is a similar pattern to Coloboma mice (M. D. Jones et al., 2001a, 2001b).

Although not as widely studied as many other transgenic models, the PI3Kγ KO mouse has shown much broader and consistent ADHD-like effects, including reversal by

ADHD medications, than many other more well-studied models. Consequently, it should perhaps be prioritized for further study.

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3.2.9 39,XY*O mouse / steroid sulfatase (STS) KO mice

39,XY*O mice have an end to end fusion of the X and Y chromosomes resulting in the deletion of the Sts gene (Odorisio, Rodriguez, Evans, Clarke, & Burgoyne, 1998).

39,XY*O mice are hyperactive (Trent et al., 2013; Trent, Dennehy, et al., 2012). 39,XY*O mice were tested in a 1 Choice Serial Reaction Time test in which the intertrial interval was manipulated to examine attention and impulsivity (Trent et al., 2013). There was no evidence of deficits in either domain, but 39,XY*O mice did show evidence of increased motivation (a greater number of nose poke responses), which was consistent with a significantly higher breakpoint on a progressive ratio schedule in another study (Trent,

Cassano, et al., 2012). In the Trent et al. (2013) study hippocampal serotonin levels were elevated, and these increases correlated with increased nose poke responses. These increases were spread across all parts of the task, and like control mice the majority of nose pokes occurred during stimulus presentation. There were more “perseverative” nose-pokes during the period just after stimulus presentation in these mice. A more extensive neurochemical analysis found that there were regionally-dependent changes in serotonin and norepinephrine function in these mice (Trent, Cassano, et al., 2012), elevated serotonin in the hippocampus and striatum, and reduced levels of the norepinephrine metabolite MOPEG in the striatum. No differences were found in the frontal cortex.

Although the work with these mice is not extensive, the findings do warrant further investigation of other ADHD-like phenotypes, as well as validation using drugs that treat ADHD.

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3.2.10 γ aminobutyric acid transporter 1 (GAT1) KO mice

GAT1 KO mice have motor abnormalities, but no differences in locomotion in the open field were observed, although other aspects of exploratory behavior were altered

(Chiu et al., 2005). Most subsequent studies were conducted with a different strain of

GAT1 KO mice (Cai et al., 2006). Despite changes in gross motor behavior, other aspects of the behavior of GAT1 KO mice have been compared to ADHD and schizophrenia (P.

Yang, Cai, Cai, Fei, & Liu, 2013; Yu et al., 2013). Perhaps because of the broad importance of GABA in so many brain functions, GAT1 KO mice exhibit changes in a wide variety of functions, including aggression, anxiety and depression-like behavior

(Liu, Cai, et al., 2007; Liu, Liu, et al., 2007). Despite the initial open field finding, other studies have found that both GAT1 +/- and -/- mice are hyperactive (Liu, Cai, et al.,

2007; Yu et al., 2013), and hyperactivity is observed during adolescence in GAT -/- mice

(Yu et al., 2013). This hyperactivity was not selectively reduced by treatment with amphetamine or methylphenidate in GAT1 -/- mice (P. Yang et al., 2013). GAT1 -/- mice did show attenuated locomotor stimulation to amphetamine, perhaps because of their initially elevated levels of activity, but all genotypes showed reductions in locomotion under at least some dose conditions for both drugs in this study. GAT1 -/- mice showed only reductions in activity after methylphenidate, but some doses of methylphenidate also reduced activity in GAT1 +/+ and GAT1 +/- mice. These effects in all genotypes make the effects in GAT1 -/- mice difficult to interpret unambiguously.

GAT1 -/- mice show broad impairments in learning that include impairments in the Morris Water Maze and Passive Avoidance (Shi et al., 2012). Curiously, GAT1 +/- mice show improvement in learning in the same tests. This demonstrates an important 67

shortcoming in many studies of knockout strains, that often focus on homozygous mice, and ignore heterozygous mice, even when the heterozygous condition might better represent the range of variation in gene/protein expression commonly seen in humans.

GAT1 -/- mice were trained in a runway task that was motivated by food (P. Yang et al.,

2013). Learning of the task was normal, but the performance of these mice was impaired by the addition of a distractor. GAT1 +/- mice were not impaired. GAT1 -/- mice also have deficits in PPI, which was linked to schizophrenia-like deficits (Yu et al., 2013), but may be more broadly linked to frontostriatal dysfunction characteristic of a number of disorders, including ADHD. These types of deficits occur in other mouse models of

ADHD. That study also showed that GAT -/- mice also had deficits in working memory and latent inhibition, but the latter deficits may have been confounded by impairments in fear conditioning in these mice.

Yu et al. (2013) associated the behavioral deficits in GAT -/- mice with increased

GABA inhibitory tone in the prefrontal cortex. Indeed, picrotoxin reversed several behavioral outcomes in GAT -/- mice. This mechanism is interesting since many other

ADHD models also affect glutamatergic activity in the prefrontal cortex, although perhaps via other mechanisms. Nonetheless, this may represent a certain degree of convergence on causative mechanisms in ADHD.

3.2.11 Genetic modifications of nicotinic acetylcholine receptors (nAChRs)

Given much of the data discussed in this review regarding the effects of nicotine in ADHD and of acetylcholine in cognition and attention, it might be expected that genetic modifications of nAChRs might provide useful models to study ADHD, or 68

perhaps ADHD-like deficits in cognition and attention. Indeed, gene knockouts for various nicotinic and muscarinic acetylcholine receptors have been shown to have effects on learning, memory, attention and other aspects of cognition (for review see L. Zhang

(2006) and E. D. Levin (2012)).

nAChR β2 KO mice have been suggested to be a potential model of ADHD

(Granon & Changeux, 2006), in part based upon the observation of slight locomotor hyperactivity. This was not found in a second study (Guillem et al., 2011). However, these mice had improved spatial learning (Granon, Faure, & Changeux, 2003). In the 5-

CSRTT nAChR β2 KO mice had increased omissions, but all other measures of performance, attention and impulsivity were unaffected (Guillem et al., 2011). Similar impairments are also observed in α7 nACHR KO mice in the 5-CCPT (Young et al.,

2007), particularly under conditions of increased attentional demand.

There is strong evidence for a role of genetic variation in disorders of cognition that include ADHD (Sinkus et al., 2015), as well as contributing to smoking in ADHD

(McClernon & Kollins, 2008). Thus, it is surprising that more mouse genetic models have not linked cholinergic system genes to ADHD-like phenotypes.

3.2.12 Fragile X mental retardation 1 (FXMR1) KO mice

The fragile X mental retardation 1 (FXMR1) KO mouse is an animal model of

ADHD that shows both autistic- and ADHD-like deficits consistent with those that occur in Fragile X syndrome. These mice emit more premature responses in tests of sustained attention indicative of impulsivity, and under some conditions have reduced accuracy and greater numbers of omissions indicative of impaired attention, including in the presence 69

of a distracting stimulus (Moon et al., 2006). These types of deficits were not confirmed in the 5-CSRTT, but FXMR1 KO mice did show perseverative impairments (Kramvis,

Mansvelder, Loos, & Meredith, 2013). FXMR1 KO mice also show evidence of impaired executive function (Dickson et al., 2013). FXMR1 KO mice are hyperactive (Kramvis et al., 2013; Wrenn, Heitzer, Roth, Nawrocki, & Valdovinos, 2015), but methylphenidate does not reduce this hyperactivity (Wrenn et al., 2015). A novel object recognition deficit is reversed by amphetamine in these mice however (Ventura, Pascucci, Catania,

Musumeci, & Puglisi-Allegra, 2004)

Although there are clear ADHD-like deficits in this model, it is really a model for fragile X syndrome, and as such includes a wide range of other deficits, including social behavior deficits (McNaughton et al., 2008), a broader range of cognitive deficits than are perhaps characteristic of ADHD (Boda, Mendez, Boury-Jamot, Magara, & Muller, 2014;

Krueger, Osterweil, Chen, Tye, & Bear, 2011; Vinueza Veloz et al., 2012), and a number of other phenotypes that are characteristic of this disorder. Consequently, the presentation of ADHD phenotypes in this model are complicated by these other factors, which may make comparisons to ADHD outside of the Fragile X context difficult. Indeed, medication of ADHD symptoms in Fragile X patients is complicated by these other conditions (Tranfaglia, 2011)

4.0 Conclusion

Based on the current review of the existing literature, it appears that the most well-validated genetic models of ADHD-like deficits involve reductions in DAT function, in particular DAT KO mice. It is also almost certainly the case that deficits in 70

these models overlap several frontostriatal disorders that share overlapping symptomatology, and perhaps underlying causal factors, with ADHD. This is not surprising since disruption of DAT function produces alterations in frontostriatal function that is also seen in other conditions. Indeed, disruption of frontostriatal function is a recurring theme in many of the models discussed here. In any case, the DAT KO model has sufficient predictive validity that it should be an effective tool for the evaluation of novel therapeutics. It is possible that the DAT +/- model may also be effective for this purpose, but the effects of this genetic manipulation are more subtle than the homozygous deletion, so it will be essential to further validate this model using more sophisticated approaches to assess the functional domains of attention, executive function and inhibitory control.

In part based upon human genetic findings, many other genetic models have been developed that may model ADHD symptoms. However, most of these models have been studied much less extensively than the DAT genetic models, and for many of these models the suggestion that they may model aspects of ADHD is based solely or primarily on the observation of hyperactivity, or the ability of psychostimulant drugs to reduce that hyperactivity. In evaluating the potential validity of these models, it will be essential to explore behavior representing a wider range of ADHD symptoms. This is especially true since symptom profiles in ADHD patients differ widely among individuals, between males and females, between adults and adolescents, and many ADHD patients do not even present with hyperactivity. Additionally, most of these models look for ADHD-like phenotypes resulting from single gene manipulations, which is not characteristic of

ADHD generally. The polygenic nature of the disorder is something that the field will 71

need to contend with at some point. Finally, except for a small number of strains, the vast majority of genetic manipulations intended to model ADHD have used gene knockout approaches, and usually homozygous knockouts as well. It will be important to try to more closely model the sort of variation observed in humans with ADHD, whether that involves more intermediate reductions in gene expression than are observed in homozygous knockout animals or knock-in strategies that express known human variants.

72

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Chapter 3

The effects of reduced dopamine transporter expression on the sex-dependent effects of isolation-rearing

Yasir Saber a,b, F. Scott Halla

a Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH, USA; b Ninevah College of medicine, Ninevah university, Mosul, Iraq

Manuscript in Preparation for submission to Pharmacology, Biochemistry and Behavior.

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Abstract

Background: Homozygous dopamine transporter knockout (DAT -/-) mice exhibit behavioral phenotypes characteristic of ADHD, but the behavior of heterozygous DAT knockout (DAT +/-) mice is only mildly affected. Social isolation of mice after weaning

(isolation-rearing) also produces a variety of pathological behavioral phenotypes associated with enhanced dopaminergic function. This raises the possibility that isolation rearing of DAT +/- mice may produce greater effects, more similar to DAT -/- mice. An additional possibility is that the magnitude of effects in DAT +/- mice, with or without isolation rearing, may also be affected by sex.

Methods: Male and female, DAT +/+ and DAT +/- mice were reared from weaning in isolation (singly) or in socially (3-4 / cage) for 8 weeks. Behavior was then assessed in 3 models that have been shown to be sensitive to the effects of DAT gene deletion and social isolation: spontaneous and amphetamine-stimulated locomotion, the cliff avoidance reaction (CAR) and prepulse-inhibition of acoustic startle (PPI).

Results: Male, but not female DAT +/- mice were initially hyperactive. Amphetamine produced locomotor decreasing effects in both male and female DAT +/- mice. CAR was impaired in both male and female DAT +/- mice, but these effects were ameliorated by social isolation. PPI was not affected by genotype or isolation rearing in males. In DAT

+/+ female mice social isolation impaired PPI, but this was not observed in DAT +/- females.

Conclusions: Contrary to expectation, the effects of social isolation and heterozygous deletion of DAT on locomotion, CAR and PPI were not found to be additive. In many instances isolation rearing normalized the effects of heterozygous DAT gene knockout or 116

vice versa. Many of these effects were also sex-dependent. The current findings do not support the notion that a combined DAT +/-, isolation rearing model will improve upon previous models of ADHD.

Keywords: Social isolation, dopamine transporter, mice, prepulse inhibition, cliff avoidance reaction, locomotor activity, amphetamine

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1.0 Introduction

Homozygous dopamine transporter knockout (DAT -/-) mice have been suggested to model aspects of ADHD, and it has recently been stated that the DAT -/- model is

“currently the most validated model of ADHD” (de la Pena et al., 2018). Indeed, that review suggested that the DAT -/- model had strong face, and predictive validity, but only partial construct validity. In terms of face validity DAT -/- mice have been shown to exhibit all of the core symptoms of ADHD. The most prominent of these was pronounce hyperactivity in a novel environment (Fox, Panessiti, Hall, Uhl, & Murphy, 2013; Giros,

Jaber, Jones, Wightman, & Caron, 1996; Ralph, Paulus, Fumagalli, Caron, & Geyer,

2001; Sora et al., 2001; Sora et al., 1998), which is characterized by reduced habituation and perseveration of initial exploratory tendencies. DAT -/- mice also have shown consistent deficits in prepulse inhibition of acoustic startle (PPI) (Arime, Kasahara, Hall,

Uhl, & Sora, 2012; Ralph et al., 2001; Wong et al., 2012; Wong, Sze, Chang, Lee, &

Zhang, 2015; Yamashita et al., 2006; Yamashita et al., 2013), a preattentional sensorimotor gating reflex. Deficits in this model have been interpreted to reflect potential attentional deficits in DAT -/- mice, although this is not really a test of attention per se. DAT -/- mice have also been shown to have impairments in the cliff avoidance reaction (CAR) (Yamashita et al., 2013), that involve falling from a small, elevated platform. DAT +/+ mice are very cautious about approaching the edges of the platform

(cliff avoidance), and very rarely fall. By contrast, the majority of DAT -/- fall in this task, which either reflects impulsivity, or impaired risk assessment. The risk assessment

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interpretation may reflect impaired decision-making or executive function in DAT -/- mice, which both are consistent with cognitive aspects of ADHD.

It would also be expected that an animal model of ADHD-like impairments would produce deficits in learning tasks, although it is likely that such deficits would not result from deficits in learning per se, but rather would result indirectly from other ADHD-like deficits, such as hyperactivity, impulsivity, or attentional impairment. DAT -/- mice have been repeatedly shown to exhibit impaired learning in a variety of tasks, including the radial arm maze (Gainetdinov et al., 1999), novel object recognition (Wong et al., 2012;

Wong et al., 2015), social transmission of food preferences (Wong et al., 2012; Wong et al., 2015), spontaneous alternation (Li, Arime, Hall, Uhl, & Sora, 2010), and the Morris

Water Maze (Morice et al., 2007). Interesting, the deficits in the Morris Water Maze, in which several variations were tested, did not seem to depend upon task difficulty, but rather upon other aspects of the experimental environment. The tendency of DAT -/- to persist in initial response tendencies may account for these results. In a novel environment this results in persistent exploratory responses, which are thought to underlie reduced marble burying in DAT -/- in the marble burying task (Fox et al., 2013). DAT -/- mice essentially ignore the marbles, persisting for the entire duration of the task at exploring the test cage. In the MWM the DAT KO mice persist in the normal initial response tendency, which to try to escape the maze, persistently exploring the walls of the maze, attempting to escape (Hall, unpublished observations).

Many of the behavioral deficits observed in the DAT -/- model have been shown to be reversed by drugs that are effective treatments for ADHD – evidence of the predictive validity of the model. Locomotor hyperactivity in DAT KO -/- mice is 119

reversed by drugs that are effective ADHD treatments, including amphetamine and methylphenidate (Gainetdinov et al., 1999). PPI deficits in DAT -/- mice are also reversed by methylphenidate (Yamashita et al., 2006). The selective NET inhibitor nisoxetine, similar to the effective ADHD treatment atomoxetine, also reverses PPI impairments in DAT -/- mice (Yamashita et al., 2006). DAT -/- induced CAR are also ameliorated by both methylphenidate and nisoxetine (Yamashita et al., 2013). High smoking rates in ADHD patients (van Amsterdam, van der Velde, Schulte, & van den

Brink, 2018) are an indication that nicotine is used for self-medication for attentional and cognitive deficits by ADHD patients (for discussion of this hypothesis see Hall, Arime,

Saber, and Sora (2016). Nicotine has also been shown to reduce both hyperactivity and

PPI deficits in DAT -/- mice (Uchiumi et al., 2013).

In evaluating the construct validity of the DAT -/- model de la Pena et al. (2018) pointed out that the evidence for reduced DAT function, genetic or otherwise, in ADHD is inconsistent at best. There certainly is evidence for dopaminergic and DAT changes in

ADHD however, including genetic differences (Faraone & Larsson, 2019; Hansen et al.,

2014; Mergy et al., 2014). Genetic cases of extreme DAT dysfunction in humans is rare, and associated with Dopamine Transporter Deficiency Syndrome, which results in early onset Parkinsonism as well as ADHD-like symptoms. Parkinsonian-like changes in dopamine function has also been described to result from compensatory changes in DAT -

/- mice (Jaber et al., 1999). Of relevance to construct validity for the DAT -/- mouse as a model of ADHD there is evidence of corticostriatal dysfunctions that may be similar to those observed in ADHD that involve noradrenergic function (Biederman & Spencer,

1999). The first evidence of this was that DAT -/- mice, although having extracellular 120

dopamine levels in the striatum that are increased by 10-fold, the prefrontal cortex is relatively unaffected (Shen et al., 2004; Xu et al., 2009), resulting in an imbalance in striatal and prefrontal dopamine function. This imbalance leads to altered corticostriatal connectivity and function that is seen in imaging, structural and anatomical studies

(Berlanga et al., 2011; Costa et al., 2006; Cyr et al., 2003; Cyr, Caron, Johnson, &

Laakso, 2005; Zhang et al., 2010). Although these findings suggest that DAT -/- mice have some degree of construct validity as a model of ADHD, despite the extreme nature of the complete knockout, it raises the question of whether DAT +/- mice might provide a better model.

The perturbations in dopamine function in DAT +/- mice are smaller and less consistent than those observed in DAT -/- mice, including only about a doubling of extracellular dopamine levels (Giros et al., 1996; Jones et al., 1998). Initial studies did not find behavioral differences in DAT +/- mice (Giros et al., 1996; Sora et al., 1998), but a slight hyperactivity can be observed under some conditions (Dluzen, Ji, & McDermott,

2010; Hall, Itokawa, et al., 2014; Mereu et al., 2017; Spielewoy et al., 2000). PPI deficits are not observed in DAT +/- mice (Mereu et al., 2017; Ralph et al., 2001). Cognitive deficits have also been observed in DAT +/- mice under some conditions (Cybulska-

Klosowicz, Dabrowska, Niedzielec, Zakrzewska, & Rozycka, 2017; Cybulska-

Klosowicz, Laczkowska, Zakrzewska, & Kaliszewska, 2017; Franca et al., 2016; Mereu et al., 2017).

Although some ADHD-like phenotypes are observed in DAT +/- mice, the effects are much weaker than in DAT -/- mice. One possibility for improving the DAT +/- model would be the addition of another factor that might potentiate the effects of the partial 121

DAT deletion. One possibility is social isolation at weaning (isolation rearing) which has been shown to produce perturbations in dopamine systems ((Hall, Wilkinson, et al.,

1998); for review see (Hall, 1998; Hall & Perona, 2012)). The behavioral consequences of isolation rearing include mild hyperactivity (Hall, Huang, Fong, Pert, & Linnoila,

1998; Hall, Wilkinson, et al., 1998; Wilkinson et al., 1994) and impairments in PPI

(Wilkinson et al., 1994), among other effects. Consequently, a series of studies was conducted to determine if isolation rearing of DAT +/- mice would potentiate their

ADHD-like phenotypes. Furthermore, in part because of the reported sex differences in

ADHD (Rhee & Waldman, 2004), the potential contribution of sex to these effects was also studied.

2.0 Materials and methods.

2.1 Subjects

Male and female, DAT +/+ and DAT +/- mice were used in these experiments

(N=10 per experimental condition). These mice were from the DAT KO line originally developed by Ichiro Sora (Sora et al., 1998) from a colony established at the University of Toledo in 2015. Mice were produced by heterozygous DAT KO crosses. The mice were housed in a standard light cycle 12/12 hrs (lights on at 7am), standard temperature

(20-22 °C), and standard humidity (40-60%). At weaning mice tissue samples for genotyping were taken by ear punches, and ear tags attached for identification. Mice were genotyped by PCR for DAT genotypes as described in previous publications (Perona et al., 2008). All experiments were conducted in accordance with all applicable guidelines

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for animal research, including those of the Association for Assessment and Accreditation of Laboratory Animal Care and the National Institutes of Health (NIH, USA) under protocols approved by the University of Toledo Institutional Animal Care and Use

Committee.

2.2 Rearing Conditions

At weaning male and female mice were divided into to same-sex rearing groups: isolation-reared and socially-reared. Mice in the isolation-reared group were housed singly under standard laboratory conditions (water and food ad libitum, 12/12–h light/dark, and humidity and temperature controlled) housing conditions. Mice in the socially-reared group were housed in groups of 5 mice. All mice remained in these housing conditions for 8 weeks prior to testing in adulthood.

2.3 Locomotor activity

Locomotion was assessed in four white opaque Plexiglas chambers (50×50×50 cm) over 3 hours using an Anymaze video tracking system (Stoelting, Wood Dale, IL).

Mice were placed gently in the middle of the testing chamber individually under dim red light in a quiet room. The boxes were cleaned with acetic acid solution between tests. The dependent measure determined using the Anymaze system was distance (m). After one hour of testing, subjects were gently removed and injected with sterile saline (10 mL/kg), placed back into the chamber, and locomotor activity was assessed for another hour. At the end of the second hour the subjects were gently removed, injected with d-

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amphetamine (2 mg/kg IP; 10 mL/kg; dissolved in sterile saline). D-amphetamine was purchased from Sigma Aldrich (St. Louis, MO).

2.4 Prepulse inhibition (PPI)

PPI was assessed using SR-LAB startle response system (San Diego Instruments,

San Diego, CA). Briefly, mice are placed in a small cylindrical enclosure that is attached to a piezoelectric accelerometer in a sound attenuated box. A high frequency speaker is positioned behind the enclosure. Before testing, mice are habituated to the box for 10 min for two days. The test begins with 5 min acclimation to the apparatus during which 65 dB background noise is emitted from the speakers. The mice are then subjected to 6 pulse- only trials (e.g. startle; consisting of a 40 ms intense acoustic stimuli (120dB)), after which they are pseudo-randomly exposed to 10 trials each of 5 trial types: no-stimulus, four prepulse-pulse intensities (3, 6, 9 and 112 dB above background 20 ms prepulses that precede the 40 ms 120 dB pulse by 100 ms), and pulse-only trials. The session is concluded by 6 pulse-only trials. The piezoelectric accelerometer measures downward force created by the mice as they startle, which is the outcome measure. The maximum startle produced in response to the 120-dB pulse is normally reduced by a relatively weaker non-startling prepulse that precedes the pulse by a short time. The entire session lasts for 25 min. PPI is calculated by the following equation: 푃푃퐼 = ((푝푢푙푠푒 −

(푝푟푒푝푢푠푙푒 + 푝푢푙푠푒))/푝푢푙푠푒) ∗100.

2.5 Cliff Avoidance Reaction (CAR)

The CAR apparatus consists of a circular platform with a diameter of 20 cm elevated 50 cm above the floor. DAT +/+ mice normally avoid approaching the edge of 124

the platform, and if they do, they are very cautious, rarely falling. On the other hand,

DAT -/- mice approach the edge in a dangerous manner, even sometimes trying to climb underneath the platform, so that over 50% of them fall from the platform (Yamashita et al., 2013). In this test, mice are placed individually on the platform for 60 min. Their behavior was recorded digitally for the first 10 min of the test and subsequently evaluated by an observer blind to genotype falls, as well as the latency to fall. If mice fell, they were returned to the platform. The primary outcome measure was the percentage of mice in each experimental group falling.

2.6 Statistical Analysis

Each portion of the locomotor data (habituation, locomotion after saline injection, and locomotion after amphetamine injection) were analyzed separately using analysis of variance (ANOVA). Data from male and female mice were analyzed separately. Because differences in basal locomotion and locomotion after saline injection were observed, amphetamine data was also calculated as difference from the final saline locomotion value to account for differing basal levels of activity prior to amphetamine injection. The data were evaluated with the between-subjects factor of GENOTYPE (DAT +/+ vs. DAT

+/-), and REARING CONDITION (socially-reared vs. isolation-reared), and the within- subjects factors of TIME (6 10-min time bins for each portion of the data: habituation, saline and amphetamine). Data are presented as mean ± SEM. All analyses were performed using SPSS version 22. statistical software. The alpha value was set at p<0.05.

Bonferroni’s test was applied for post hoc means comparisons.

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Statistical analysis of PPI data (startle magnitude and % PPI) was performed by

ANOVA with the between-subjects factor of GENOTYPE (DAT +/+ vs. DAT +/-) and

REARING CONDITION (socially-reared vs. isolation-reared), and the within-subjects factor of PREPULSE VALUE for %PPI (4, 8, and 16 dB). Bonferroni’s test was applied for post hoc means comparisons. All analyses were performed by using the SPSS statistical package v. 22. The alpha value was set at p<0.05. Data for PPI are presented as mean ± SEM.

CAR performance was assessed in terms of the proportion of mice successfully remaining on the platform for the whole 60 min test (% CAR). These proportions were analyzed using χ2 tests. All analyses were performed by using the SPSS statistical package v. 22. The alpha value was set at p<0.05. Data for startle and PPI are presented as mean ± SEM.

3.0 Results

3.1 Locomotor activity

The locomotor activity of male mice during the initial habituation period (0-60 min) are shown in Fig. 1A. Locomotor activity habituated slightly over the course of the habituation period, although this differed across the treatment groups. A significant overall effect of TIME was confirmed in the ANOVA (F (3.0, 94.5) = 4.9, P<0.05). DAT

+/- mice were more active than DAT +/+ mice throughout the testing period, which was confirmed by a significant effect of GENOTYPE in the ANOVA (F (1,31) = 23.5,

P<0.001). The locomotor activity of socially reared male DAT +/- mice was higher initially but habituated over the course of the habituation period. By contrast, the 126

locomotor activity of isolation-reared male DAT +/+ mice was not as elevated initially but did not habituate. Consequently, post hoc comparisons showed that the socially isolated male DAT +/- mice had higher locomotor activity compared to socially-reared

DAT +/+ mice during the first 2 time-bins, while male DAT +/- mice had elevated activity compared to socially-reared DAT +/+ male mice in all time-bins after the first 10 min (P<0.001 vs. social control). Consistent with this pattern, while the main factor

REARING CONDITION was not significant (F (1,31) = 0.3, NS), the REARING

CONDITION x TIME interaction was significant (F (3.0, 94.0) = 9.5, P<0.0001).

Figure 1.

Male A B

Habituation phase Saline phase ✱ ✱✱✱ 60 ✱ ✱ ✱ 60 ✱✱

✱✱ DAT+/+Social ✱ DAT+/+Social s s DAT+/+ Isolated

DAT+/+ Isolated r

r

e

e

t

t e

e 40 DAT+/- Social 40 DAT+/- Social

m

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n DAT+/- Isolated DAT+/- Isolated

i

i

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e

c

c n

n 20 20

a

a

t

t

s

s

i

i

D D

0 0 10 20 30 40 50 60 10 20 30 40 50 60 Time in min. Time in min. C D

Amphetamine phase Comparison between amphetamine and saline

60 80

DAT+/+Social ✱ DAT+/+Social e

n ✱

i 60 l

s DAT+/+ Isolated ✱ DAT+/+ Isolated

r

e

e s

t ✱ a

e 40 DAT+/- Social 40 DAT+/- Social

b

m

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n DAT+/- Isolated DAT+/- Isolated i

o 20

r

f

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e n

20 c 0

a

n

t

e

s

r

i e

D -20

f

f i

0 D -40 10 20 30 40 50 60 10 20 30 40 50 60 Time in min. Time in min. Figure 1. Locomotion in male DAT +/+, DAT +/-, socially-reared (social) and isolation-reared mice. (A) Habituation, (B) Saline, (C) Amphetamine, and (D) Difference between amphetamine and saline. * p<0.05; ** p<0.01; *** p<0.001.

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Moreover, the REARING CONDITION x GENOTYPE x TIME interaction was also statistically significant (F (3.0, 94.5) = 4.6, P<0.01).

After saline injection (Fig. 1B), the differences in locomotor activity observed during the habituation session persisted at Figure 2 the level observed at the end of the A Male Saline ✱✱✱ ✱ habituation session throughout this period 50 ✱✱

✱✱ ✱ ✱ DAT+/+ s

r 40 DAT+/-

e t

so that the effect of TIME was not e m

30

n

i

e

c 20

n a

significant in the ANOVA (F (5,113.6) = t s

i 10 D

0 0.7, NS). There were also no significant 10 20 30 40 50 60 Time in min. interactions of TIME with GENOTYPE or B Amphetamine phase

60

REARING CONDITION (F (5,113.6) = DAT+/+ s

r DAT+/-

e t

e 40 m

0.5, NS; F (5,113.6) = 0.7, NS;

n

i

e c

n 20

a

t s

respectively). However, male DAT +/- i D

0 mice continued to have elevated 10 20 30 40 50 60 Time in min. locomotion resulting in a significant main C Comparison between amphetamine and saline

60 effect of GENOTYPE (F (1,31) =16.7, ✱✱ DAT+/+

s ✱✱ r DAT+/-

e ✱✱ t 40 ✱✱

e ✱✱ m

✱✱

P<0.001; Fig. 1B and 3A). Rearing n i

20

e

c

n

a t

s 0

condition did not affect locomotor activity i D

-20 in male DAT +/- mice so that there was no 10 20 30 40 50 60 Time in min. significant interaction between Figure 2. Locomotor activity in male DAT +/+ and DAT +/- mice: A) after saline GENOTYPE and REARING injection; B) after amphetamine injection; C) the difference between activity after CONDITION (F (1,31) = 0.653, NS). To amphetamine and the final level of activity under saline. * p<0.05; ** p<0.01; *** further illustrate the effect of genotype the p<0.001. 128

data from male mice were averaged without respect to rearing condition for the saline phase (Fig. 2A). Male DAT +/- mice were clearly hyperactive and were significantly different from DAT +/+ mice at all time-points.

Because of these baseline differences in activity a more detailed consideration of the effects of amphetamine in male mice must be given (Fig. 1C/D, and 2B/C). Firstly, an

Figure 3 Female A B Habituation Saline phase

80 80

DAT+/+Social DAT+/+Social s

s DAT+/+ Isolated DAT+/+ Isolated r

r 60 60

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e

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a t t 20

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0 0 10 20 30 40 50 60 10 20 30 40 50 60 Time in min. Time in min.

D C

Amphetamine phase Comparison between amphetamine and saline ✱✱ ✱✱ ✱✱✱ 80 60 ✱✱✱

✱ ✱✱ DAT+/+Social ✱✱ ✱✱✱ DAT+/+Social

e n

i ✱✱ l

s ✱✱ DAT+/+ Isolated 40 DAT+/+ Isolated r

60 e ✱✱

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n t

20 e

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e

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f

f i 0 D -40 10 20 30 40 50 60 10 20 30 40 50 60 Time in min. Time in min. Figure 3. Locomotion in female DAT +/+, DAT +/-, socially-reared (social) and isolation-reared mice. (A) Habituation, (B) Saline, (C) Amphetamine, and (D) Difference between amphetamine and saline. * p<0.05; ** p<0.01; *** p<0.001.

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additional ANOVA was conducted to Figure 4 compare the amphetamine treatment in male Female A mice to activity during saline treatment Saline DAT 30

DAT+/+ s

r DAT+/-

e t e 20

using TREATMENT (saline vs. m

n

i

e c

n 10

a

t

s i amphetamine) and TIME as within-subjects D 0 10 20 30 40 50 60 factors and REARING CONDITION and Time in min.

GENOTYPE as between subjects factor. B Firstly, comparing the raw activity data after Amphetamine phase 80

✱✱✱ ✱✱✱ DAT+/+ s

r ✱✱✱ DAT+/- e

t 60 ✱✱ ✱✱

amphetamine (Figs. 1C and 2B) to the saline e

m

n ✱ i

40

e

c

n a data (Figs. 1B and 2A) it is clear that there t

s 20

i D

0 was an increase in activity in male DAT +/+ 10 20 30 40 50 60 Time in min. mice but not the activity of male DAT +/- C Comparison between amphetamine and saline ✱✱✱✱

60 ✱✱✱✱ e

n DAT+/+ i mice. This was confirmed by a significant l ✱✱✱✱ ✱✱✱✱ e 40 s ✱✱✱✱ DAT+/-

a ✱✱✱

b

m 20 o

interaction between TREATMENT x r

f

e 0

c

n e

r -20

e

f f

GENOTYPE (F (1,31) = 10.0, P<0.01). i

D -40 10 20 30 40 50 60 There was also a significant effect of TIME Time in min. but not TIME x TREATMENT (F (2.9, Figure 4. locomotor activity of different DAT genotype female mice A) locomotor 88.2) = 3.1, p<0.05; F (2.9, 88.2) = 2.7, NS; effect of saline injection B) Amphetamine 2mg/kg injection effect on different DAT respectively). No other effects were genotype female mice C) the difference between amphetamine and saline significant in the ANOVA. This overall locomotor activity. * p<0.05, **p<0.01, ***p<0.0001, ****p<0.00001. pattern is even clearer when the data is calculated as a difference score (Figs. 1D and 2C), taking into account differences in baseline activity between groups. When the data were expressed in this way, there is 130

clearly a locomotor stimulatory effect of amphetamine in male DAT +/+ mice compared to DAT +/- mice, which showed little effect; indeed, activity was slightly reduced by amphetamine. The locomotor stimulatory effect in male DAT +/+ mice peaks at 20 min, and then reduces after that, consequently there is a significant effect of TIME (F (3.1,

94.5) = 4.9, p<0.0001). The main effect of GENOTYPE was significant in the ANOVA

(F (1,31) = 16.1, P<0.001), but not REARING CONDITION (F (1,31) = 0.3, NS).

However, the TIME x REARING CONDITION (F (3.1, 94.5) = 9.5, p<0.0001) and

TIME x GENOTYPE x REARING CONDITION (F (3.1, 94.5) = 4.6, p<0.05), which seems to reflect the small reduction in the effects of amphetamine in isolation-reared

DAT +/+ male mice, but not DAT +/- male mice. No other effects were significant in the

ANOVA.

In female mice, much more limited effects of both GENOTYPE and REARING

CONDITION were observed. Only slight habituation in locomotor activity was observed across the first two hours of testing, including after saline injections. There was a significant effect of TIME in the first hour of habituation reflecting small decreases in locomotion over the time of testing (F (5, 175) = 8.3, P<0.001; Fig. 3A). This differed slightly across groups, so there was a significant interaction between REARING

CONDITION and TIME (F (5, 175) = 4.5, P<0.01), primarily due to a small initial difference in activity between female isolation-reared DAT +/+ mice and female socially- reared DAT +/+ mice. All other effects were not significant. There were no significant effects of any factors after saline treatment (Fig. 3B). The data is again shown without respect to the rearing groups to better show the effects of genotype (Fig.4). In Fig 4A, the female DAT +/- mice had a slight increase in activity, but this was not significant. 131

Amphetamine clearly increased locomotor activity in female DAT +/+ mice, but not in female DAT +/- mice, and indeed produced clear reductions in locomotion compared to the levels seen at the end of the saline phase (Figs. 3C and 4B). This pattern of effects was confirmed by significant effects of Figure 5 TIME (F (5, 175) = 8.9, p<0.01) and Male Mice A Social B Isolated GENOTYPE (F (1, 35) = 20.3, 100 100 ✱ ✱✱ 80 80

% 60

% 60

p<0.001) but not REARING

R

R

A

A C 40 C 40

CONDITION (F (1, 35) = 0.7, NS) in 20 20

0 0 the ANOVA, as well as a significant DAT+/+ DAT+/- DAT+/+ DAT+/-

GENOTYPE x TIME interaction (F D C DAT +/+ DAT +/-

100 100 (5,175) = 6.3, p<0.0001). None of the ✱✱✱✱

80 ns 80 %

60 % 60

other effects were significant. When the

R

R

A

A C 40 C 40 data were expressed as the difference 20 20

0 0 from the final saline measurement the Social Isolated Social Isolated effects of amphetamine were even more Figure 5. % CAR success (not falling in 60 min) in male (A) socially-reared mice, (B) clear (Figs. 3D and 4C): amphetamine isolation-reared mice, (C) DAT +/+ mice, and (D) DAT +/- mice. **p<0.01, increased locomotion in female DAT ****p<0.0001

+/+ mice, but reduced locomotion in female DAT +/- mice. This was confirmed by a significant main effect of GENOTYPE in the ANOVA (F (1, 35) = 36.7, p<0.001). There was a trend for female isolation-reared DAT +/+ mice to have reduced locomotion compared to their socially-reared counterparts, but this was not significant, and the effects of REARING CONDITION and REARING CONDITION x TIME were not significant (F (1, 35) = 0.7, NS; F (5, 175) = 1.1, NS; respectively). 132

3.2 Cliff Avoidance Reaction (CAR)

CAR responses were affected in a complex fashion by genotype, sex, and isolation-rearing. To represent these complex interactions different comparisons of the data are shown in Figures 5-7. Figure 6 CAR responses in male mice are Female mice A B Social Isolated ✱✱ shown in Fig. 5. Firstly, comparing Fig. 100 ✱✱✱✱ 100 80 5 to Fig 6, it is clear the male DAT +/+ 80

% 60

% 60

R

R

A A

C 40 mice had impaired CAR compared to C 40

20 20 female DAT +/+ mice. CAR success 0 0 DAT+/+ DAT+/- DAT+/+ DAT+/- was defined by the number of mice that

C D did not fall (CAR %) during the 60 min DAT+/+ DAT+/- ✱✱✱✱ ✱✱✱✱ 100 100 80 test. Separate comparisons are shown for 80

% 60

% 60

R

R

A A socially-reared and isolation-reared DAT C 40

C 40

20 20 +/+ and DAT +/- male mice. In male 0 0 Social Isolated Social Isolated mice, socially-reared DAT +/- mice fell Figure 6. % CAR success (not falling in 60 min) in off the platform more often than female (A) socially-reared mice, (B) isolation- reared mice, (C) DAT +/+ mice, and (D) DAT +/- socially-reared DAT +/+ mice (Fig. 5A; mice. **p<0.01, ****p<0.0001 x2 (1) = 8.9, p<0.01). This effect was eliminated in isolation-reared mice (Fig. 5B).

Indeed, male isolation-reared DAT +/- mice fell less often than DAT +/+ mice (x2 (1) =

5.1, p<0.05). Isolation rearing did not affect CAR in male DAT +/+ mice (Fig. 5C; x2 (1)

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= 0.3, NS), but increased CAR % in female DAT +/- mice (Fig. 5D; x2 (1) = 21.2, p<0.0001).

CAR responses in female mice are shown in Fig. 6. CAR success was defined by the number of mice that did not fall (CAR %) during the 60 min test. Separate comparisons are shown for socially-reared and isolation-reared DAT +/+ and DAT +/- female mice. CAR performance in female socially-reared DAT +/+ female mice was superior to that observed in DAT+/- female mice (x2 (1) = 18.01, p< 0.0001, Figure 5A).

Socially isolated DAT+/+ female mice showed a significant higher CAR performance in compare DAT+/- female mice ((x2 (1) = 10.53, p< 0.01, figure 5B). Social isolation significantly increased CAR performance relative to socially reared in DAT+/+ female mice ((x2 (1) =19.78, p< 0.0001, figure 5C). Even in DAT+/- female mice, social isolation significantly increased CAR performance ((x2 (1) = 32.14, p < 0.0001, figure

5D).

The effects of sex are shown in each of the experimental groups in Fig. 7.

Socially-reared male DAT +/+ mice had significantly lower CAR performance compared with socially-reared female DAT +/+ mice (x2 (1) = 8.2, p<0.01, Fig. 7A). CAR performance was impaired in both sexes of socially-reared DAT +/- mice (Fig. 7B), and was not significantly different (x2 (1) = 2.4, NS). Isolation-reared male DAT +/+ mice had significantly reduced CAR performance compared with isolation-reared female DAT

+/- mice (Fig. 7C; x2 (1) = 50.0, p< 0.0001). Surprisingly, isolation-rearing improved

CAR performance in DAT +/- mice, although males again had impaired CAR performance compared to females (Fig. 7D; x2 (1) = 7.8, p < 0.0001).

134

In summary, impairments in CAR performance were observed in DAT +/- mice.

In addition, male mice performed poorly in this task compared to female mice.

Surprisingly, isolation rearing improved performance in the CAR task in all groups except male DAT +/+ mice. Consequently, the effects of partial DAT depletion were reversed by isolation-rearing.

3.3 Pre-pulse Inhibition of Acoustic Startle (PPI)

PPI was affected by sex, genotype, and isolation-rearing in a complex manner that Figure 7 interacted with prepulse intensity. A Social DAT+/+ B Social DAT+/-

In male mice there was a significant 100 ✱✱ 100

80 80 ns

% 60

effect of PREPULSE INTENSITY (F % 60

R

R

A A

C 40

C 40

(3,78) =124.4, p<0.001; Fig. 8A), and 20 20

0 0 female Male there was a trend toward an interaction female Male between DAT Genotype, Rearing C D Isolated DAT+/+ Isolated DAT +/- ✱✱✱✱ 100 ✱✱ condition and Pre-pulse intensity. Male 100 80 80

% 60

% 60

isolation-reared DAT +/- mice had R

R

A A

C 40

C 40 slightly reduced PPI at lower stimulus 20 20 0 0 Female Male Female Male intensities. This trend was not observed Figure 7. % CAR success (not falling in 60 in male DAT +/+ mice. In any case the min) comparisons of male and female mice: (A) socially-reared DAT mice, (B) main effects of REARING socially-reared DAT +/- mice, (C) Isolation-reared DAT +/+ mice, and (D) CONDITION and GENOTYPE, as well Isolation-reared DAT +/- mice. **p<0.01, ****p<0.0001 as the REARING CONDITION x

135

GENOTYPE interaction, were not significant (F (1,26) = 3.0, NS; F (1,26) =1.3, NS; F

(1,26) = 2.2, NS, respectively).

Figure 8

A B

90 Female 90 Male social 70 70 DAT+/ + 50 * * 50

* %PPI %PPI 30 30 * 10 10

-10 68 71 74 77 -10 68 71 74 77 prepulse intensity dB Prepulse intensity dB Figure 8. rearing condition and DAT genotypes on Prepulse inhibition A) the effect of DAT genotype and rearing condition on PPI in female mice B) the effect of DAT genotype and rearing condition on PPI in male mice. *p<0.05 In female mice, there was a significant main effect of PREPULSE INTENSITY (F (3,93)

= 48.3, P < 0.001; Fig. 8B), reflecting the increase in PPI with higher prepulse intensities.

There was a significant main effect of REARING CONDITION, but not GENOTYPE, in female mice (F (1,31) = 14.7, P < 0.001; (F (1,31) = 0.1, NS, respectively). Isolation- rearing impaired PPI, but these reductions were only lower in DAT +/+ mice in post hoc comparisons (p<0.05). In the ANOVA, the interactions between PREPULSE

INTENSITY and REARING CONDITION, and GENOTYPE and PREPULSE

INTENSITY were not significant (F (3,93) = 1.3, NS; F (3,93) = 0.5, NS, respectively).

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4.0 Discussion

Contrary to expectation, rather than potentiating behavioral effects of heterozygous DAT deletion, isolation-rearing reversed many of those effects, or in some cases, effects that appeared to result from isolation-rearing were not observed in DAT +/- mice. Additionally, the effects of DAT genotype and isolation rearing on behavior were highly dependent on sex, a factor which has not previously been considered. It is not simply that such effects were observed only in one sex, but rather that different effects were observed in each sex.

Consistent with some previous observations (Dluzen et al., 2010; Hall, Itokawa, et al., 2014; Mereu et al., 2017; Spielewoy et al., 2000), modest locomotor hyperactivity was observed in DAT +/- mice. However, this modest hyperactivity was observed in male mice, but not female mice, except for the very first time-bin. Female mice also exhibited no habituation over the period of testing which might account for some of these results. It must also be noted that unlike previous locomotor studies in DAT KO mice

(Hall, Itokawa, et al., 2014; Hall, Sora, Hen, & Uhl, 2014; Sora et al., 2001; Sora et al.,

1998), the current study used a video system placed above open field boxes to monitor locomotion rather than an infrared-beam system in an enclosed sound-attenuating box.

The current study was conducted under red-light conditions in a quiet, empty room, but there still appeared to be less overall habituation of locomotor activity than in previous studies. Isolation-rearing attenuated the slight hyperactivity observed in male DAT +/- mice, which was seen as a reduction of this hyperactivity towards the end of the second hour of testing, after the saline injection.

137

Amphetamine and other stimulants have been previously observed to reduce locomotor hyperactivity in DAT -/- mice rather than producing the locomotor stimulant effects characteristic of DAT +/+ mice (Gainetdinov et al., 1999; Hall, Sora, et al., 2014).

Although the degree of hyperactivity was not as great as is observed in DAT -/- mice, and in fact in female mice there was no hyperactivity by the time of the amphetamine injections, amphetamine-induced hyperactivity was not observed, and slightly reduced locomotion was observed instead. Although this was a somewhat high dose, it did induce locomotor hyperactivity in DAT +/+ mice, and the reductions in locomotion did not appear to result from stereotypical behavior in DAT +/- mice.

Previous studies in isolation reared rats have shown that isolation-rearing induces deficits in PPI (Wilkinson et al., 1994). These effects are also seen in both 129 and

C57BL/6J mouse strains (Varty, Powell, Lehmann-Masten, Buell, & Geyer, 2006), the strains used to generate the DAT KO strain studied here (Sora et al., 1998). Previous studies in DAT -/- mice have also shown deficits in PPI (Arime et al., 2012; Ralph et al.,

2001; Yamashita et al., 2006; Yamashita et al., 2013), but not DAT +/- mice (Mereu et al., 2017; Ralph et al., 2001). There were some interactions between rearing condition and DAT genotype on PPI. In males there was trend toward a reduction in PPI in isolated

DAT +/- mice, but there was no difference between socially-reared and isolation-reared

DAT +/+ mice. By contrast, in socially-reared female mice PPI was impaired in DAT +/- mice compared to DAT +/+ mice, but there was no effect of genotype in isolation-reared female mice. This would seem to indicate that for some phenotypes there is a complex interaction between sex, DAT genotype, and isolation-rearing. In male mice deficits were only observed in isolation-reared DAT +/- mice, which is an additive effect that is in 138

accordance with our initial prediction. However, that effect was small, and there was only a trend toward statistical significance. In socially-reared female mice there was an effect of DAT genotype (DAT +/- less than DAT +/+), but this effect was eliminated in DAT

+/- mice.

Similar complex interactions of DAT genotype and rearing condition with sex were observed in the CAR test. Isolation rearing had not been previously assessed in

CAR, nor had DAT +/- mice, although DAT -/- mice have been shown to have CAR deficits (Yamashita et al., 2013). CAR deficits were shown in both male and female DAT

+/- mice compared to DAT +/+ mice. In both cases isolation rearing reversed those deficits, contrary to our initial prediction. The nature of these CAR effects are open to question, as was discussed in the original publication of CAR deficits in DAT -/- mice

(Yamashita et al., 2013). The first possible explanation is increased impulsivity in DAT

+/- mice. However, since falling from the platform often occurred after an extended period of time on the platform, it seems that falling is not an impulsive act, but rather just a poor choice. The mice exhibit risky behavior, placing themselves far over the platform, often with their front fee on the underside, risking a fall. Thus, it would appear that this deficit is a deficit of risk assessment or some other aspect of executive function. This would still be consistent with an ADHD-like deficit, although deficits of this nature are certainly seen in other frontostriatal disorders as well (for discussion see Young,

Winstanley, Brady, and Hall (2017).

DAT -/- mice have been repeatedly suggested to be a valid animal model of

ADHD (Arime, Kubo, & Sora, 2011; de la Pena et al., 2018; Gainetdinov et al., 1999;

Yamashita et al., 2013). However, de la Pena et al. (2018) pointed out that the construct 139

validity of the DAT -/- model is weak for several reasons. Firstly, the evidence for reduced DAT function, genetic or otherwise, in ADHD is inconsistent at best, although there is certainly evidence for perturbations in DAT and DAT function in ADHD (Faraone

& Larsson, 2019; Hansen et al., 2014; Mergy et al., 2014). One of the most obvious weaknesses is that genetic cases of extreme DAT dysfunction in humans are rare, and associated with non-ADHD like impairments, in addition to ADHD-like impairments. At the same time there is evidence of corticostriatal dysfunctions in DAT -/- mice that are similar to those observed in ADHD (Biederman & Spencer, 1999). This includes an imbalance in cortical and subcortical dopamine function in DAT -/- mice (Shen et al.,

2004; Xu et al., 2009), that affects corticostriatal connectivity and function (Berlanga et al., 2011; Costa et al., 2006; Cyr et al., 2003; Cyr et al., 2005; Zhang et al., 2010). Thus, despite weaknesses, the DAT -/- mouse does show certain mechanistic similarities to the changes thought to underlie ADHD. Nonetheless, this raises the question of whether DAT

+/- mice might provide a better model of ADHD.

However, both the alterations in dopamine function and behavior in DAT +/- mice are smaller than those observed in DAT -/- mice (Giros et al., 1996; Jones et al.,

1998). Indeed, some studies show no locomotor differences in DAT +/- mice (Giros et al., 1996; Sora et al., 1998), although others have found a slight hyperactivity can under some conditions (Dluzen et al., 2010; Hall, Itokawa, et al., 2014; Mereu et al., 2017;

Spielewoy et al., 2000). PPI deficits are not observed in DAT +/- mice (Mereu et al.,

2017; Ralph et al., 2001), but some cognitive deficits have been observed (Cybulska-

Klosowicz, Dabrowska, et al., 2017; Cybulska-Klosowicz, Laczkowska, et al., 2017;

Franca et al., 2016; Mereu et al., 2017). Based on the supposition that the effects of 140

heterozygous DAT deletion were too mild to produce a strong ADHD-like phenotype, we proposed that the combination of a heterozygous DAT deletion together with isolation from weaning would produce a stronger ADHD-like phenotype. This was hypothesized because isolation-rearing produces many similar phenotypes that are associated with a hyperdopaminergic state that may also involve reduced corticostriatal function (Hall,

1998; Hall & Perona, 2012).

Contrary to expectation, the combination of partially reduced DAT function and isolation-rearing did not produce greater effects, but rather, in most instances, appeared to counteract each other. Nonetheless, some of the first evidence is provided here that the heterozygous DAT deletion alone produces behavioral phenotypes characteristic of

ADHD, including locomotor hyperactivity and impaired CAR. Moreover, paradoxical locomotor inhibitory effects of amphetamine were also observed in DAT +/- mice. This data encourages further examination of the DAT +/- model as a potential model of

ADHD that may have greater validity that the DAT -/- model, and especially examination of more complex cognitive and attentional testing that has proved difficult to accomplish in DAT -/- mice.

141

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Chapter 4

Sex-dependent effects of the serotonin 1B receptor antagonist SB 224289 in an animal model of Attention Deficit Hyperactivity Disorder

Yasir Saber a,b, ; Raghad A. Elhag a; F. Scott Hall a a Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH, USA; b Ninevah College of

Medicine, Ninevah University, Mosul, Iraq

Manuscript in Preparation for submission to Behavioural Pharmacology.

150

Abstract

Introduction: Perturbations in dopamine signaling have been implicated in Attention

Deficit Hyperactivity Disorder (ADHD), although they likely involve developmental effects on neural circuits controlling attention, response selection, response inhibition and decision making. Homozygous DAT knockout (DAT -/-) mice model many ADHD-like deficits that include locomotor hyperactivity, impairments in the cliff avoidance reaction

(CAR), and prepulse inhibition (PPI) deficits. This study investigated whether the serotonin 1B receptor antagonist SB 224289 would reduce ADHD-like deficits in DAT -

/-, including effects in heterozygous DAT KO (DAT +/-) mice, as well as male and female mice.

Methods: Male and female DAT +/+, DAT +/- and wildtype (DAT +/+) mice were tested for the effects of 20 mg/kg SB 224289 on locomotor activity, CAR, and PPI. This dose was chosen based on previous findings showing that it reduced locomotor hyperactivity in DAT -/- mice.

Results: Male and female DAT -/- male mice were hyperactive, and female DAT +/- mice were slightly hyperactive. SB 224289 did not affect this hyperactivity. CAR was impaired in male and female DAT -/- mice. SB 224289 rescued CAR deficits in female

DAT -/- mice, but not male DAT -/- mice. Female DAT -/- female mice had PPI deficits, which were not significant in male mice. This deficit was also rescued by SB224289.

Conclusions: This study found sex-dependent deficits in CAR and PPI in DAT -/- mice. the 5HT1B antagonist SB 224289 ameliorated many of these deficits, providing additional evidence supporting this as a novel target for the treatment of ADHD.

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Keywords: Dopamine transporter, 5HT1B receptor, attention deficit hyperactivity disorder, prepulse inhibition, cliff avoidance reaction, locomotor activity, SB 224289

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Introduction

The neurotransmitter dopamine has a crucial role in many of the behavioral domains that are affected in Attention Deficit Hyperactivity Disorder (ADHD) including motor function, attention, behavioral inhibition, response selection and executive function

(Nieoullon, 2002; Sagvolden & Sergeant, 1998). These deficits are described in a slightly different manner in the Research Domain Criteria (RDoC) criteria framework (Musser &

Raiker, 2019), but the same types of deficits are described. The RDoC approach to psychiatric nosology is intended to rectify several perceived deficits in DSM approaches

(for discussion see (Young, Winstanley, Brady, & Hall, 2017). These deficits include the recurrence of particular symptoms in multiple diagnoses, symptom heterogeneity across individuals with the same diagnoses, and a general failure of DSM approaches to nosology to incorporate biological markers of disease into psychiatry nosology. Although still at quite preliminary stages this is the ultimate goal of the RDoC initiative (Kozak &

Cuthbert, 2016). Although alterations in dopamine function are thought to be involved in

ADHD, circuit level changes in occurring in prefrontal and striatal circuitry that are influenced by dopamine dynamics appear to be causally related to the disorder (Robbins,

2007), although this certainly extends to broader cortical circuitry as well (Castellanos &

Proal, 2012). These conceptions of the causal factors underlying ADHD are certain to influence how ADHD is modeled in animals.

As the etiology of ADHD is known to involve both genetic and environmental causes (Bidwell et al., 2017; Faraone & Larsson, 2019; Palladino, McNeill, Reif, &

Kittel-Schneider, 2019), animal models have focused on both aspects. The dopamine transporter (DAT) has been a particular focus of ADHD models based on evidence from 153

human genetic models (Genro et al., 2008; Grunblatt, Werling, Roth, Romanos, &

Walitza, 2019; Hong, Hwang, Lim, Kwon, & Jin, 2018; Yang et al., 2007), as well as the fact that psychomotor stimulant drugs that treat ADHD modulate dopamine function.

This does not mean that DAT polymorphisms account for ADHD in any simple manner.

The genetic causes of ADHD are highly polygenic and heterogeneous (Faraone &

Larsson, 2019), and interact with environmental factors (Palladino et al., 2019). Indeed,

DAT variation is not identified in GWAS studies of ADHD (Demontis et al., 2019), indicative of the complex nature of the underlying causality in ADHD. DAT missense mutations are observed in humans, but are quite rare (Hansen et al., 2014). Moreover, these missense mutations, although they are associated with ADHD-like symptoms, are also Parkinsonian-like motor impairments. More subtle deficits in DAT function produced by genetic mutations are also associated with ADHD, but they are also associated with Autism Spectrum Disorder, Bipolar disorder, and Dopamine Transporter

Deficiency Syndrome (DTDS; a Parkinsonian-like disorder) (Mergy et al., 2014). Indeed, the numerous mutations identified in this study are all rare, and perhaps better represent the type of genetic causality that we should expect – many routes to the same consequences.

In RDoC terms, this would suggest that we should search for common endophenotypes; in this case, perhaps similar changes in dopamine dynamics. Imaging studies in ADHD patients have sought such mechanisms, sometimes finding that striatal

DAT binding or availability is reduced (Hesse, Ballaschke, Barthel, & Sabri, 2009), but more often finding increases (Cheon et al., 2003; Krause, Dresel, Krause, la Fougere, &

Ackenheil, 2003; Spencer et al., 2007). Thus, while neither the human genetic or imaging 154

data support clear causal relationships for alterations in DAT function with ADHD, in particular reduced function, they do consistently observe differences in DA and DAT function in ADHD. Many genetic models of ADHD have focused upon altering DAT function, usually reductions in DAT function of one degree or another. Indeed, the DAT -

/- mouse has been described as “currently the most validated transgenic animal model of

ADHD” (de la Pena et al., 2018). That summary of the model emphasized both face and predictive validity of the model. Indeed, DAT -/- are hyperactive (Giros, Jaber, Jones,

Wightman, & Caron, 1996; Sora et al., 1998), and this hyperactivity is reduced by stimulant drugs that treat ADHD (Gainetdinov et al., 1999). DAT -/- mice also have deficits in pre-pulse inhibition of acoustic startle responses (PPI), a pre-attentional sensorimotor gating reflex (Ralph, Paulus, Fumagalli, Caron, & Geyer, 2001), that are also reversed by stimulant drugs that are effective ADHD treatments (Yamashita et al.,

2006). This is also true for DAT KO-induced deficits in the cliff avoidance reaction

(Yamashita et al., 2013). Nisoxetine, a norepinephrine transporter blocker with actions similar to the ADHD medication atomoxetine, also reverses PPI and CAR deficits in

DAT -/- mice (Arime, Kasahara, Hall, Uhl, & Sora, 2012; Yamashita et al., 2006;

Yamashita et al., 2013).

Although perhaps not a direct consequence of the behavioral consequences of

DAT KO, various deficits in tests of learning and cognitive function are also observed in

DAT -/- mice (Del'Guidice et al., 2014; Dzirasa et al., 2009; Gainetdinov et al., 1999; Li,

Arime, Hall, Uhl, & Sora, 2010; Morice et al., 2007; Wong et al., 2012; Wong, Sze,

Chang, Lee, & Zhang, 2015). These are likely to reflect indirect impairments in learning or cognitive performance produced by hyperactivity, impulsivity, or impaired executive 155

function/decision-making in DAT KO mice, as they do in ADHD. Cognitive deficits in one of these tasks that involve impairments in alternation and reversal learning are reduced by atomoxetine (Del'Guidice et al., 2014). Also of relevance to the predictive validity of the DAT -/- model are reductions in locomotor hyperactivity, PPI deficits and cognitive impairments by nicotine (Del'Guidice et al., 2014; Uchiumi et al., 2013; Weiss,

Nosten-Bertrand, McIntosh, Giros, & Martres, 2007; Weiss, Tzavara, et al., 2007), consistent with the high smoking rates in ADHD (van Amsterdam, van der Velde,

Schulte, & van den Brink, 2018) reflecting self-treatment.

In addition to this data demonstrating face and predictive validity for the DAT -/- model there is some evidence for construct validity as well. Although the homozygous

DAT KO produces some phenotypes that are not necessarily characteristic of ADHD, similar to what is observed in human missense mutations (Hansen et al., 2014), other evidence suggests that they show circuit-level changes that are characteristic of ADHD.

Imbalances in striatal and prefrontal dopamine function in DAT -/- mice (Shen et al.,

2004) are thought to lead to impaired frontostriatal function (Arime et al., 2012; Li et al.,

2010), involving both cellular level changes (Berlanga et al., 2011; Cyr et al., 2003; Cyr,

Caron, Johnson, & Laakso, 2005) and circuit level changes (Costa et al., 2006; Dzirasa et al., 2009; Zhang et al., 2010). Alterations in prefrontal cortex function are central to these changes in DAT -/- mice, and nisoxetine has been shown to act in the prefrontal cortex

(Arime et al., 2012), indicating that the primary site of action of drugs treating ADHD may be in the prefrontal cortex. Uchiumi et al. (2013) developed a model of the circuitry potentially underlying these effects (a modified version is shown in Fig. 1). Of particular importance, they found that the effects of nicotine could be reversed by a 5-HT1A 156

antagonist, suggesting that drugs acting at 5-HT receptors might influence DAT KO phenotypes, and by implication be useful in the treatment of ADHD. Supporting this idea,

Barr et al. (2004) found that 5-HT2A antagonists reduced PPI deficits and hyperactivity in DAT -/- mice. The 5HT1B antagonists SB 224289 has also been shown to reduce hyperactivity in DAT -/- mice (Hall, Sora, Hen, & Uhl, 2014).

Figure 1

Figure 1. Schematic illustration of the neural circuitry that may underlie the effects of nisoxetine, methylphenidate, and nicotine in DAT KO mice involving the medial prefrontal cortex (mPFC). Stimulants and methylphenidate act to increase extracellular levels of dopamine and norepinephrine in the prefrontal cortex by inhibiting NET. Nicotine acts at α7 nicotinic receptors to induce 5-HT1A receptor-dependent serotonin release. Other serotonin receptors either here, or in other parts of this circuit may influence its function. This includes 5-HT1B receptors in several portions of this circuit. Abbreviations: DAT (dopamine), GluR (glutamate receptors), LC (locus coeruleus), NE (norepinephrine), Pyr (prefrontal pyramidal neurons), VTA (ventral tegmental area), and 5-HT (serotonin).

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To further examine the potential of 5HT1B receptor antagonism as a potential

ADHD treatment, the effects of SB 224289 (20 mg/kg IP; the effective dose from the

Hall, Sora, et al. (2014) study) on DAT KO induced locomotor hyperactivity, PPI deficits and CAR impairments were studied. Additionally, the effects of heterozygous DAT deletion (DAT +/-) was also studied, as well as the effects of DAT deletion in male and female mice.

2.0 Materials and methods

2.1 Subjects

Male and female DAT +/+ mice, DAT +/-, and DAT -/- were used in these experiments (N = 8-12 per experimental condition). Testing occurred between 2 and 4 months of age. This DAT KO strain is the one originally developed by Ichiro Sora (Sora et al., 1998). A colony of these mice was established at the University of Toledo in 2016.

Mice for these experiments were produced by heterozygous crosses. The mice were housed in a standard light cycle 12/12 hrs (lights on at 7am), standard temperature (20-22

°C), and standard humidity (40-60%). At weaning mice tissue samples for genotyping were taken by ear punches, and ear tags attached for identification. Mice were genotyped by PCR for DAT genotypes as described in previous publications (Perona et al., 2008).

All mice were socially housed, 3-4 per cage, with food and water available ad libitum prior to training. All experiments were conducted in accordance with all applicable guidelines for animal research, including those of the Association for Assessment and

Accreditation of Laboratory Animal Care and the National Institutes of Health (NIH,

USA), as described in the Guide for the care and use of laboratory animals (National 158

Research Council (U.S.). Committee for the Update of the Guide for the Care and Use of

Laboratory Animals., Institute for Laboratory Animal Research (U.S.), & National

Academies Press (U.S.), 2011) under protocols approved by the University of Toledo

Institutional Animal Care and Use Committee.

2.2 Locomotor activity

Locomotion was assessed in four white opaque Plexiglas chambers (50×50×50 cm) over 3 hours using an Anymaze video tracking system (Stoelting, Wood Dale, IL).

Mice were placed gently in the middle of the testing chamber individually under dim red light in a quiet room. The boxes were cleaned with Rescue™ spray (dilute hydrogen peroxide solution) between tests. The dependent measure determined using the Anymaze system was distance (m).

Subjects were tested 3 times: first with no injection, and on the next two occasions after IP injections with vehicle or 20 mg/kg SB 224289, in a volume of 10 mL/kg. The duration of testing was 30 min. This period of testing was chosen based upon the timing of the locomotor decreasing effects observed in a previous study (Hall, Sora, et al., 2014).

2.3 Prepulse inhibition (PPI)

PPI was assessed using SR-LAB startle response system (San Diego Instruments,

San Diego, CA). Briefly, mice are placed in a small cylindrical enclosure that is attached to a piezoelectric accelerometer in a sound attenuated box. A high frequency speaker is

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positioned behind the enclosure. Before testing, mice are habituated to the box for 10 min for two days. The test begins with 5 min acclimation to the apparatus during which 65 dB background noise is emitted from the speakers. The mice are then subjected to 6 pulse- only trials (e.g. startle; consisting of a 40 ms intense acoustic stimuli (120dB)), after which they are pseudo-randomly exposed to 10 trials each of 5 trial types: no-stimulus, four prepulse-pulse intensities (3, 6, 9, and 12 dB above background 20 ms prepulses that precede the 40 ms 120 dB pulse by 100 ms), and pulse-only trials. The session is concluded by 6 pulse-only trials. The piezoelectric accelerometer measures downward force created by the mice as they startle, which is the outcome measure. The maximum startle produced in response to the 120-dB pulse is normally reduced by a relatively weaker non-startling prepulse that precedes the pulse by a short time. The entire session lasts for 25 min. PPI is calculated by the following equation: 푃푃퐼 = ((푝푢푙푠푒 −

(푝푟푒푝푢푠푙푒 + 푝푢푙푠푒))/푝푢푙푠푒) ∗100.

Subjects were tested 2 times: the sequence of treatment was pseudo-random for each GENOTYPE group ether IP injections with vehicle or 20 mg/kg SB 224289, in a volume of 10 mL/kg.

2.4 Cliff avoidance reaction (CAR)

The CAR apparatus consists of a circular platform with a diameter of 20 cm elevated 50 cm above the floor. DAT +/+ mice normally avoid approaching the edge of the platform, and if they do, they are very cautious, rarely falling. On the other hand,

DAT -/- mice approach the edge in a dangerous manner, even sometimes trying to climb underneath the platform, so that over 50% of them fall from the platform (Yamashita et 160

al., 2013). In this test, mice are placed individually on the platform for 60 min. The primary outcome measure was the percentage of mice in each experimental group falling.

Subjects were tested 2 times: the sequence of treatment was pseudo-random for each

GENOTYPE group ether IP injections with vehicle or 20 mg/kg SB 224289, in a volume of 10 mL/kg.

2.5 Drug Treatments

Treatments were administered during the challenge sessions in a counterbalanced manner with at least 48 hours between treatments. Drugs were administered 5 minutes prior to testing. The 5-HT1B antagonist SB 224289 (1'-methyl-5-[[2'-methyl-4'-(5- methyl-1,2, 4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6,7-tetrahydro-spiro[furo[2,3- f]indole-3,4'-piperidine]; Santa Cruz Biotech) was dissolved in 10% β hydroxypropyl cyclodextrin in distilled water (vehicle), and tested at a doses of mg/kg IP in a volume of

10 mL/kg. This dose was chosen based upon a previous study (Hall, Sora, et al., 2014).

2.6 Statistical Analysis

Basal locomotor data was analyzed separately from the effects of SB 22428 using analysis of variance (ANOVA). Data from male and female mice were analyzed separately. The data were evaluated with the between-subjects factor of Genotype (DAT

+/+, DAT +/- and DAT -/-), and the within-subjects factors of Drug (SB 224289 vs. vehicle) and Time (six 5-min time bins) where appropriate. Data are presented as mean ±

SEM. All analyses were performed using SPSS version 22 statistical software. The alpha value was set at p<0.05. Bonferroni’s test was applied for post hoc means comparisons. 161

Statistical analysis of PPI data (startle magnitude and % PPI) was performed by

ANOVA with the between-subjects factor of Genotype (DAT +/+, DAT +/- and DAT -/-), and the within-subjects factors of Drug (SB 224289 vs. vehicle) and Pre-pulse value for %PPI (4, 8, and 16 dB), where appropriate. Bonferroni’s test was applied for post hoc means comparisons. All analyses were performed by using the SPSS statistical package v.

22. The alpha value was set at p<0.05. Data for startle and PPI are presented as mean ±

SEM.

Statistical analysis of observational data in the CAR test (stretch attend postures, head dips, fecal boli, the number of falls, and the latency to fall) was performed by

ANOVA with the between-subjects factor of Genotype (DAT +/+, DAT +/- and DAT -/-) and the within-subjects factor of Drug (SB 224289 vs. vehicle). Bonferroni’s test was applied for post hoc means comparisons. The proportion of mice falling in each group was analyzed using χ2 analysis. All analyses were performed by using the SPSS statistical package v. 22. The alpha value was set at p<0.05. Data for startle and PPI are presented as mean ± SEM.

3.0 Results

3.1 Locomotor activity

Locomotor activity of male and female DAT+/+, DAT+/-, and DAT-/- mice are represented in Figs. 2A and 2B under basal conditions and in Figs. 3A-F after SB 224289.

Locomotor activity of male DAT+/+, DAT+/-, and DAT-/- mice was elevated compared to both DAT +/+ and DAT +/- mice. The testing period in this experiment was somewhat short (30 min), so only limited habituation was observed in DAT +/+ and DAT +/- mice.

However, DAT -/- mice did not habituate over that time period, and indeed the activity of 162

these mice increased over the testing Figure 2 period. These patterns were confirmed by a significant main effect of Genotype in a repeated measures ANOVA (F (2, 27) =

46.0, p<0.0001) and a significant

Genotype x Time interaction (F (10, 135)

= 6.1, p<0.0001). Post hoc comparisons showed that male DAT-/- mice had higher locomotor activity in comparison with male DAT +/+ mice at all time-points

(p<0.00001). The locomotor activity of Figure 2. Locomotor activity in a novel male DAT+/- mice did not differ from environment in DAT +/+, DAT +/- and DAT -/- mice. A) Male. B) Female. male DAT +/+ mice. ***p<0.001 vs. vehicle, ****P<0.0001 vs. vehicle. A similar overall pattern was observed for female mice (Fig. 2B). Similar to male DAT-/- mice, female DAT-/- mice were very hyperactive, and this elevated activity did not habituate over time. Indeed, there was again a slight increase in activity over the testing period. By contrast a slight habituation was observed in female DAT +/+ mice. In female DAT +/- the habituation was slightly attenuated, and female DAT +/- mice were more active than female DAT

+/+ mice. This pattern of effects was confirmed by a significant main effect of Genotype in a repeated measures ANOVA (F (2, 26) = 25.4, p<0.0001) and a significant Genotype x Time interaction (F (10, 130) = 7.8, p< 0.0001). Post hoc comparisons showed that female DAT-/- mice had higher locomotor activity than female DAT +/+ mice at all time- 163

points (p<0.0001). In addition, the activity of female DAT +/- mice was also higher in comparison to female DAT +/+ mice at all time-points (p<0.0001).

Figure 3 A D

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Figure 3. The effects of SB 224289 (20 mg/kg IP) on locomotor activity on male (A/B/C) and female (D/E/F), DAT +/+ (A/D), DAT +/- (B/E), and DAT -/- (C/F) mice compared to placebo (vehicle). As was observed during the habituation session, mal DAT -/- mice were 164

hyperactive during subsequent locomotor testing (Fig. 3A-C). An ANOVA for the locomotor activity after SB 224289 or vehicle administration in male mice revealed a significant main effect of Genotype (F (2,27) = 63.8, P<0.0001), as well as a significant

Time and Genotype interaction, reflecting the lack of habituation in DAT -/- mice (F

(5.7,76.3) = 9.7, P<0.0001). Indeed, the activity of DAT -/- mice increased over the test period (Fig. 3C). Post hoc comparisons showed that male DAT -/- had higher locomotor activity than male DAT +/- and DAT +/+ mice (P<0.001 at all time-points). No differences were observed between male DAT +/- and DAT +/+ mice. In male DAT +/- mice there was a slight trend for SB 224289 to reduce locomotor activity, but ANOVA did not find a significant main effect of SB 224289 Treatment (F (1,27) = 0.6, NS), nor were any of the interactions with SB 224289 Treatment significant.

The effects of SB 224289 on locomotor activity in male DAT +/+, DAT +/-, and

DAT -/- mice are represented in Figs. 6A, 6B, and 6C, respectively. Consistent with the overall ANOVA separate analyses of the effects of SB 224289 did not find significant effects of SB 224289 in any genotype of male mice: DAT +/+: F (1, 14) = 0.14, NS; DAT

+/-: F (1, 20) = 0.43, NS; or DAT -/-: F (1, 18) = 0.54, NS.

The effects of SB 224289 on locomotor activity in female DAT +/+, DAT +/-, and DAT -/- mice are represented in Figs. 3D, 3E, and 3F, respectively. As was found for the habituation condition female DAT KO mice were hyperactive, as was confirmed by a significant main effect of Genotype in the ANOVA (F (2,26) = 31.9, P<0.0001). Post hoc comparisons confirmed that the locomotor activity of female DAT -/- mice was greatly elevated compared to the locomotor activity of female DAT +/+ and DAT +/- mice

(P<0.001). Indeed, rather than habituating, as was seen in female DAT +/+ and DAT +/- 165

mice, this activity increased over Figure 4 the period of testing in female A Male

80 DAT -/- mice, which confirmed ✱ DAT +/+

60 ✱✱✱ DAT +/- by a significant Time x Genotype DAT-/-

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0 (3.4,44.1) = 3.5, p<0.05). SB -20 68 71 74 77 224289 did not affect locomotor Prepulse intensity (dB) activity in female mice, although B Female 80 again there was a slight trend for 60 ✱✱

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I P DAT -/- mice as the sessions P 20 0 progressed. Nonetheless, the -20 68 71 74 77 effect of SB 224289 Treatment Prepusle intensity (dB) Figure 4. PPI (% inhibition) in male (A) and female was not significant in the (B), DAT +/+, DAT +/-, and DAT -/- mice at 4 prepulse intensities (68, 71, 74 and 77 dB; 3, 6, 9 and ANOVA (F (1,26) = 2.5, NS) nor 12 dB above the 65-dB background. *p<0.05, **p<0.01, ***p<0.001. were the Treatment x Genotype

(F (2,26) = 1.6, NS) or Treatment x Genotype x Time (F (1.8,46.4) = 2.4, NS) interactions significant. Separate analysis of the data for each of the genotypes separately

(the data presented in Figs. 3D, 3E and 3F) also did not find any significant effects of SB

224289 treatment in female mice: DAT +/+: F (1, 6) = 0.36, NS; DAT +/-: F (1, 12) =

0.77, NS; or DAT -/- (F (1, 16) = 1.1, NS).

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3.2 PPI

DAT KO produced impairments in baseline PPI that were observed in both sexes, but were only significantly different from DAT +/+ mice in female DAT -/- mice. PPI in male DAT +/+, DAT +/-, and DAT -/- mice is represented in Fig. 4A. PPI (expressed as percent inhibition) increased with PPI intensity (F (2.3, 57.7) = 36.7, p<0.0001). There was a reduction in PPI in male DAT -/- mice, but primarily in comparison to male DAT

+/- mice, not male DAT +/+ mice. Thus, there was a significant main effect of Genotype in male mice (F (2, 25) = 6.5, p<0.01). Post hoc comparisons showed that PPI was significantly higher in male DAT +/- mice in comparison to male DAT -/- mice at the 68- and 71-dB pre-pulse intensities (p< 0.001 and p<0.05 respectively).

Baseline prepulse inhibition in female DAT +/+, DAT +/-, and DAT -/- mice is represented in Fig. 4B. PPI increased with prepulse intensity, as was shown by a main effect of Prepulse Intensity in the ANOVA (F (2.0, 45.2) = 16.2, p<0.0001). PPI was reduced at the lower prepulse intensities in female DAT -/- mice. Thus, although the main effect of Genotype in the ANOVA was not significant (F (2, 23) = 2.0, NS), there was significant interaction between DAT Genotype and Prepulse Intensity (F (6, 69) = 3.2, p<

0.001). Post hoc comparisons showed that PPI was lower in female DAT -/- mice in comparison to DAT +/+ at the lowest prepulse intensity, 68 dB (p<0.05).

The effects of SB 224289 treatment on PPI are shown in Fig. 5 for male and female DAT +/+, DAT +/-, and DAT -/- mice. The data for male DAT +/+, DAT +/-, and

DAT -/- mice are represented in Figs. 5A, 5B, and 5C, respectively. PPI increased with prepulse intensity in male mice of all genotypes (F (2.1, 54.4) = 29.6, P<0001). PPI was

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reduced in male DAT -/- mice, but primarily in comparison to male DAT +/- mice. Thus, there was a significant effect of Genotype (F (2, 26) = 5.631, P<0.01), and post hoc comparisons showed that male DAT+/- mice had higher PPI compared to both male DAT

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+/+ and DAT -/- mice (P<0.01, and P<0.0001, respectively). The SB224289 treatment main factor effect was not significant (F (1,26) = 3.5,NS), nor was the interaction between Genotype and Prepulse Intensity was not significant (F (6, 78) = 2.0, NS).

The data for female DAT +/+, DAT +/-, and DAT -/- mice are shown in Figs. 5D,

5E, and 5F, respectively. PPI was reduced in female DAT -/- mice at the lowest prepulse intensity (68 dB) compared to female DAT +/+ mice. This reduction in PPI was not observed after treatment with SB 224289. No effects of genotype, or drug treatment, were apparent at higher prepulse values. This pattern of effects was confirmed by the results of the ANOVA. Although the main effects of Genotype (F (2,23) = 2.5, NS) and Drug treatment (F (1,23) = 0.8, NS) were not significant, the interaction between Genotype and

Drug treatment was significant (F (6, 69) = 3.2, P<0.05). Post hoc comparisons showed that there was a significant difference in PPI in female DAT -/- mice after treatment with

SB 224289 compared to vehicle (p<0.05).

3.3 CAR

Substantial differences in baseline CAR performance were observed between genotypes. These data are shown in Fig. 6, which shows comparisons between male and female mice (6A and 6B, respectively). In male mice, there were no differences in CAR between DAT +/+ and DAT +/- mice, but DAT -/- mice had greatly reduced CAR compared to both male DAT +/+ (χ2(1) = 115.0, p<0.0001) and male DAT +/- (χ2(1) =

115.0, p<0.0001) mice. In female mice (Fig. 6B) there were also substantial differences in CAR performance between genotypes. There was a slight, but significant reduction in

CAR performance in female DAT +/- mice compared to female DAT +/+ mice (χ 2(1) = 169

6.2, p<0.05). Like male mice, CAR performance was substantially reduced in female

DAT -/- mice, although the reduction was not as large as that seen in male DAT -/- mice.

This reduction in CAR performance was significantly different in female DAT -/- mice compared to female DAT +/+ mice (χ 2(1) = 40.5, p<0.0001) and female DAT+/- mice

(χ2(1) = 23.8, p<0.001).

The effects of SB 224289 on CAR performance in male and female, DAT +/+,

DAT +/-, and DAT -/- mice are shown in Fig. 7. In male DAT +/+ mice CAR Figure 6 B A Male Female

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0.001, Fig. 7A). CAR performance was also slightly impaired by SB 224289 in male

DAT +/- mice (χ2(1) = 9.5, p<0.01, Fig. 7B). As was seen at baseline, CAR performance in male DAT -/- mice was greatly reduced after vehicle (placebo) treatment, but CAR performance was non-improved by SB 224289 treatment (χ2(1) = 1.9, NS, Fig. 7C). Fig.

7 shows the effects of SB 224289 on CAR performance in separate comparisons for each sex and genotype group. In male DAT +/+ mice CAR performance was significantly 170

decreased after SB224289 treatment Figure 7

(χ2(1) = 28.6, p < 0.001, Fig. 7A).

CAR performance was also slightly impaired by SB 224289 in male DAT

+/- mice (χ2(1) = 9.5, p<0.01, Fig. 7B).

As was seen at baseline, CAR performance in male DAT -/- mice was greatly reduced after vehicle

(placebo) treatment, but CAR performance was not improved by SB

224289 treatment (χ2(1) = 1.9, NS,

Fig. 7C). CAR performance was not affected by SB 224289 in either female DAT +/+ (χ2(1) = 0.0, NS) or

DAT +/- (χ2(1) = 0.0, NS, Fig. 7E) mice. CAR performance was substantially impaired in female Figure 7. CAR performance in male (A/B/C) and female (D/E/F), DAT +/+ (A/D), DAT +/- (B/E) DAT -/- mice and this impairment and DAT -/- (C/E) mice after treatment with placebo (vehicle) or SB 224289 (20 mg/kg IP). was substantially ameliorated by SB Significant differences between treatment groups: **p<0.01, ***p<0.001. 224289 treatment (χ 2(1) = 18.3, p<0.001, Fig. 7F).

171

4.0 Discussion

The data presented here replicated previous finding in DAT KO mice, and showed that some small effects could also be observed in DAT +/- mice under some conditions.

The effects of DAT KO were also shown to be sex-dependent. In the current study, previously identified deficits in DAT KO mice were replicated, and additionally the effects of sex and heterozygous DAT deletion were examined. Both male and female

DAT -/- mice were hyperactive compared with DAT +/+ mice. A slight hyperactivity was also observed in female DAT +/- mice. Small deficits in PPI were also observed in DAT -

/- mice, but not DAT +/- mice, but these were only significant in female DAT -/- mice. In the CAR test deficits were observed in both male and female mice, but these deficits were greater in male mice. There was also evidence for amelioration of some of these deficits by the 5HT1B antagonist SB 224289 under some conditions, but, again, these effects were sex-dependent.

The present study confirmed findings of hyperkinesia in DAT-/- mice, which has been shown many times (Barr et al., 2004; Del'Guidice et al., 2014; Gainetdinov et al.,

1999; Giros et al., 1996). As in previous studies, these effects were quite profound, and were also see in terms of reduced locomotor habituation. The magnitude of the hyperactivity was similar in male and female DAT -/- mice; however, although male

DAT +/- mice were not different from male DAT +/+ mice, female DAT +/- mice were slightly more active than female DAT +/+ mice. A previous study did find slightly increased locomotion in DAT +/- mice, but this study did not examine sex (Hall, Itokawa, et al., 2014). SB224289 treatment in this study did not alleviate locomotor hyperactivity in DAT -/- mice as was seen in a previous study by Hall, Sora, et al. (2014), nor did it 172

affect the slight hyperactivity observed in female DAT +/- mice. The previous study found that SB 224289 reduced locomotor activity in DAT -/- mice at a dose of 20 mg/kg

IP. There were several differences between that study and this one, however, including the duration of testing (60 vs. 30 min) and testing conditions. In the previous study locomotion was assessed in a completely closed apparatus, a photobeam locomotor testing box contained within a sound-attenuating chamber. The current testing was done in an open field under red light conditions with a video monitoring of activity. Indeed, under the previous conditions there was far greater habituation of locomotor activity (in

DAT +/+ mice) than was observed under the present circumstances, indicating that the mice were responding differently to the environment. The present test only looked at one dose of SB 224289 as well. Although this was the dose that produced significant differences in the previous study, it will be essential in future studies to examine a wider range of doses, and perhaps to test for a longer period of time. Although the duration of testing was also chosen based upon the timing of the peak effects in the previous study, the testing circumstances may have altered this interaction. In any case, although no significant effects of SB 224289 were found on DAT KO-induced hyperactivity, significant effects were found on other DAT KO-induced behavioral impairments in the present study.

PPI deficits have been repeatedly found in DAT -/- mice (Arime et al., 2012;

Ralph et al., 2001; Wong et al., 2012; Wong et al., 2015; Yamashita et al., 2006). PPI deficits were also found in the present study, although they were not as robust as previous studies. The reason for this difference is not necessarily clear, although this study did include DAT +/- mice in the analysis. No deficits were found in DAT +/- mice; indeed, 173

under some conditions PPI appeared to be slightly enhanced in DAT +/- mice.

Significant, pre-pulse intensity dependent reductions in PPI were observed in female

DAT -/- mice, but not male DAT -/- mice, although these mice did show the same trend.

Previous studies have shown that PPI deficits in DAT KO mice can be reversed by drugs that are effective treatments for ADHD (Yamashita et al., 2006), as well as nicotine

(Uchiumi et al., 2013), and the selective NET blocker nisoxetine (Arime et al., 2012;

Yamashita et al., 2006), which is similar to the effective ADHD medication atomoxetine.

Although the present results did not find large deficits in PPI, they did show that SB

224289 would reverse these deficits when they did occur, at low pre-pulse intensities in female DAT -/- mice.

Yamashita et al. (2013) found that DAT -/- mice also have deficits in CAR, which could be interpreted either as a deficit in inhibitory control or decision making/risk assessment, either of which have potential relevance to ADHD. Moreover, these deficits could be ameliorated by either methylphenidate or nisoxetine, like PPI deficits in DAT

KO mice. The present results showed that these deficits were not really seen in DAT +/- mice. There was a slight deficit in female DAT +/- mice, but this effect was quite small.

The deficits in DAT -/- mice were very large, but they were much larger in male DAT -/- mice than in female DAT -/- mice. However, the differences in CAR performance were not affected by SB 224289 in male DAT -/- mice, but was improved in female DAT -/- mice by SB 224289. Since this study tested only one drug dose, it will be essential to examine other doses in male mice in future studies.

The mechanisms underlying deficits in DAT KO mice involve Imbalances in striatal and prefrontal dopamine function (Shen et al., 2004) and impaired fronto-striatal 174

function (Arime et al., 2012; Li et al., 2010). Elimination of DAT function produces both cellular (Berlanga et al., 2011; Cyr et al., 2003; Cyr et al., 2005) and circuit level changes

(Costa et al., 2006; Dzirasa et al., 2009; Zhang et al., 2010) that likely underlie the behavioral differences in DAT -/- mice. The prefrontal cortex is at the center of these changes, and the effects of nisoxetine have been linked to actions in the prefrontal cortex using intracerebral injections of nisoxetine, which reduce PPI deficits when injected into the prefrontal cortex, but not when injected into the nucleus accumbens (Arime et al.,

2012). The model developed by Uchiumi et al. (2013) (Fig. 1) suggests that serotonin manipulations might also modulate the altered activity of frontostriatal circuits in DAT -/- mice. Studies had already shown alterations in serotonergic function were important for the maintenance of cocaine reinforcement in DAT -/- mice (Hall et al., 2002; Sora et al.,

2001). Moreover, the first study to address this question suggested that serotonergic effects of locomotor stimulants mediated the locomotor decreasing effects in DAT -/- mice (Gainetdinov et al., 1999). Although a subsequent study found evidence that the effects of serotonin agonists on DAT KO-induced hyperactivity were likely artifactual

(Fox, Panessiti, Hall, Uhl, & Murphy, 2013), the same study did suggest that there were substantial perturbations in serotonin activity in DAT -/- mice. In addition, Barr et al.

(2004) found that 5-HT2A antagonists reduce PPI deficits and hyperactivity in DAT -/- mice and Hall, Sora, et al. (2014) found that the 5HT1B antagonist SB 224289 also reduces hyperactivity in DAT -/- mice. The present study did not confirm this but did find effects of SB 224289 on PPI and CAR under some conditions. The locus of these effects was not identified in the present studies, but there are certainly several likely loci where

5HT1B antagonism might affect these behaviors. Indeed, consistent with the proposed 175

circuitry, 5HT1B receptor blockade increases acetylcholine levels in the prefrontal cortex

(Hu, Wang, Stenfors, Ogren, & Kehr, 2007). Acetylcholine activates cholinergic α7 receptors on serotoninergic terminals that increase serotonin release in the prefrontal cortex which may activate serotonin 1A receptors on pyramidal neurons that project to the nucleus accumbens and or the ventral tegmental area. Projections to the ventral tegmental area modulate dopaminergic projections to the medial prefrontal cortex, which may have cognition-enhancing effects (Ago, Sakaue, Baba, & Matsuda, 2002). Although the mechanism underlying the 5HT1B-mediated effects may be different from the NET- mediated effects, Arime et al. (2012) found that nisoxetine stimulated prefrontal glutamatergic projections to the nucleus accumbens, but not the ventral tegmental area.

An alternative explanation might be that SB 224289 inhibition of presynaptic

5HT1B autoreceptors may increases serotonin neurotransmission in the prefrontal cortex.

Reduced serotonin neurotransmission, and alterations in 5HT1B receptor function in particular, has been implicated in impulsivity (Coccaro, Fanning, Phan, & Lee, 2015; da

Cunha-Bang, Hjordt, Dam, et al., 2017; da Cunha-Bang, Hjordt, Perfalk, et al., 2017).

Although there were reasons to think the effects of fluoxetine on locomotor hyperactivity in DAT-/- mice (Gainetdinov et al., 1999), were artifactual (Fox et al., 2013), they are certainly consistent with the present findings, and further investigation of fluoxetine in

DAT KO mice is certainly warranted as well. The effects of fluoxetine in DAT -/- mice are quite different from DAT +/+ mice (Hall et al., 2002; Mateo, Budygin, John, & Jones,

2004). Indeed, fluoxetine increases dopamine release in the nucleus accumbens through actions in the ventral tegmental area in DAT -/- mice, while having no effects in DAT

176

+/+ mice (Mateo et al., 2004). Fluoxetine also normalized DAT -/- impairments in PPI, although citalopram did not (Yamashita et al., 2006).

In conclusion, these studies demonstrate that while both male and female DAT -/- mice have behavioral deficits, the magnitude of some of these deficits, particularly in PPI and CAR, are sex-dependent. Few effects were observed in DAT +/- mice, but it will be worthwhile to examine these mice in more sophisticated tests of attention and cognition.

At least one study has found deficits in DAT +/- mice in tests of cognition (Mereu et al.,

2017), although it must be noted that this study found much greater effects of DAT +/- deletion on other behavioral phenotypes than other studies have found. The reason for these differences from other studies are not certain. In any case, the present results found additional evidence supporting the idea that 5HT1B antagonism reduces ADHD-like deficits in the DAT -/- model, and by implication might have beneficial effects in ADHD patients. Further studies of the effects of 5-HT1B antagonists in circumstances in which

DAT -/- and DAT +/- exhibit behavioral deficits are warranted.

177

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Chapter 5

Preclinical assessment of the Serotonin 1B receptor as a novel target for the treatment of Attention Deficit Hyperactivity Disorder

Yasir Saber ab, ; Raghad A. Elhag a; Huyen T. Tran a; Taylor M. Osting a; Chen Yu a;

Sasha H. Heeren a; F. Scott Hall a

a Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH, USA; b Ninevah College of medicine, Ninevah university, Mosul, Iraq

189

Abstract

Introduction: Dysfunctions in dopamine and dopamine transporter (DAT) function are thought to play a role in Attention Deficit Hyperactivity Disorder (ADHD). Although stimulant drugs and atomoxetine are effective treatments in some ADHD patients, alternative non-stimulant drugs are desired. Homozygous DAT knockout (DAT -/-) mice have been suggested to induce ADHD-like behavioral impairment, and that it may be used to evaluate potential novel therapeutics for the treatment of ADHD. This model was used to evaluate the potential of the serotonin 1B receptor antagonist SB 224289 as potential ADHD treatment.

Methods: Male and female DAT +/+, DAT +/-, and DAT -/- were evaluated in tests of spontaneous locomotion and the cliff avoidance reaction (CAR). The effects of atomoxetine (ATX) and SB 224289 on DAT KO-induced deficits in these tasks were examined.

Results: DAT -/- mice were hyperactive and had CAR impairments. DAT +/- mice also had CAR deficits, and were hyperactive in some groups tested. ATX reduced locomotor activity in male DAT +/- mice, and female mice of all genotypes. SB 224289 reduced locomotor activity in male DAT +/- and DAT -/- mice, but had no effects in female mice.

ATX mitigated CAR deficits in male DAT -/- and DAT +/- mice and female DAT -/- mice. SB 224289 mitigated CAR deficits in male DAT -/- and DAT +/- mice, and in female DAT -/- mice

Conclusion: DAT KO mice exhibited ADHD-like behavioral deficits that were mitigated by the established ADHD therapeutic ATX. The 5-HT1B antagonist SB 224289 also 190

ameliorated ADHD-like deficits in DAT KO mice suggesting that this may be an alternative non-stimulant approach to the treatment of ADHD.

Keywords: Dopamine transporter, cognition, impulsivity, atomoxetine, serotonin 1B receptor, SB 224289, ADHD, locomotor activity, cliff avoidance reaction

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1.0 Introduction

Attention deficit/hyperactivity disorder (ADHD) is a highly prevalent developmental neuropsychological disorder in children that affects between 5.3 and 11% of the school-age children (Lecendreux, Konofal, Cortese, & Faraone, 2015; Polanczyk,

Willcutt, Salum, Kieling, & Rohde, 2014; Visser et al., 2014). Although for many patients the symptoms are reduced later in life, in many patients, an estimated 3.4% of the adult population, ADHD symptoms persist into adulthood (Fayyad et al., 2007). ADHD incidence appears to have increased over the last decade, especially among adults and females (Fairman, Peckham, & Sclar, 2017), although this may represent more accurate diagnosis as much as an actual increase in prevalence. The negative personal and society impacts of ADHD are substantial (de Zeeuw, van Beijsterveldt, Ehli, de Geus, &

Boomsma, 2017; Fenesy, Teh, & Lee, 2019; Rietveld & Patel, 2019; Zendarski, Mensah,

Hiscock, & Sciberras, 2019; Zendarski, Sciberras, Mensah, & Hiscock, 2017), but are mitigated to some extent by effective therapeutics that include amphetamine, methylphenidate, atomoxetine and guanfacine (Chan, Fogler, & Hammerness, 2016).

However, not all individuals with ADHD are effectively treated by these mediations and side effects limit the ability to achieve effective dosing, particularly with psychostimulant drugs that produce insomnia and anorexia (Graham & Coghill, 2008), but also currently available non-stimulant drugs (Yildiz, Sismanlar, Memik, Karakaya, & Agaoglu, 2011).

There is also concern that adolescent exposure to psychostimulant drugs might increase the likelihood of developing substance use disorders later in life (Shanks et al., 2015).

Thus, it would be highly desirable to develop an alternative non-stimulant therapeutic approach to the treatment of ADHD. 192

This raises the question of how to effectively identify potential ADHD therapeutics. DAT KO mice have been suggested to be an animal model of ADHD with high predictive validity (Arime, Kubo, & Sora, 2011; de la Pena et al., 2018; Shanks et al., 2015). DAT -/- mice exhibit behavioral phenotypes consistent with all major ADHD symptoms (Diagnostic and statistical manual of mental disorders : DSM-5, 2013). These behavioral phenotypes include hyperactivity (Fox, Panessiti, Hall, Uhl, & Murphy, 2013;

Giros, Jaber, Jones, Wightman, & Caron, 1996; Ralph, Paulus, Fumagalli, Caron, &

Geyer, 2001; Sora et al., 2001; Sora et al., 1998), impaired prepulse inhibition of acoustic startle (PPI) (Arime, Kasahara, Hall, Uhl, & Sora, 2012; Ralph et al., 2001; Wong et al.,

2012; Wong, Sze, Chang, Lee, & Zhang, 2015; Yamashita et al., 2006; Yamashita et al.,

2013), impairments in the cliff avoidance reaction (CAR) (Yamashita et al., 2013), and impairments in various assessments of learning (Gainetdinov et al., 1999; Li, Arime,

Hall, Uhl, & Sora, 2010; Morice et al., 2007; Wong et al., 2012; Wong et al., 2015), that likely reflect the indirect consequences of other impairments, such as hyperactivity, impulsivity or attentional impairments. Furthermore, many of these deficits are ameliorated by effective ADHD treatments, including amphetamine and methylphenidate, including hyperactivity (Gainetdinov et al., 1999), PPI deficits

(Yamashita et al., 2006), and CAR deficits (Yamashita et al., 2013). In addition, consistent the idea that ADHD patients may smoke as a form of self-medication (van

Amsterdam, van der Velde, Schulte, & van den Brink, 2018), nicotine also reduces hyperactivity and PPI deficits in DAT -/- mice (Uchiumi et al., 2013) and improves learning deficits (Weiss, Nosten-Bertrand, McIntosh, Giros, & Martres, 2007; Weiss,

Tzavara, et al., 2007). PPI and CAR deficits in DAT -/- mice are also reversed by 193

nisoxetine (Yamashita et al., 2006; Yamashita et al., 2013), a selective NET inhibitor like atomoxetine. These data collectively suggest that the DAT KO model has a high predictive validity for ADHD treatments.

Arime et al. (2012) demonstrated that the effects of nisoxetine in DAT KO mice were due to actions on NET in the prefrontal cortex that influence the activity of corticostriatal projections. Based in part on this analysis, as well as antagonist studies,

Uchiumi et al. (2013) proposed that the actions of nicotine in DAT KO mice were mediated by actions on α7 nicotinic receptors and serotonin 1A receptors. Because of the actions of serotonin 1B receptors on serotonergic terminals (de Groote, Olivier, &

Westenberg, 2002), this was also hypothesized to be a potential approach to ameliorating

DAT KO-induced deficits (Hall, Sora, Hen, & Uhl, 2014). Serotonin 1B antagonism increases the release of acetylcholine in the prefrontal cortex (Hu, Wang, Stenfors,

Ogren, & Kehr, 2007), which is consistent with the model developed by Uchiumi et al.

(2013). Variants in the serotonin 1B receptor gene are also associated with ADHD in humans (Guimaraes et al., 2009; Hawi et al., 2002; Ickowicz et al., 2007; Quist et al.,

2003; van Rooij et al., 2015). Consistent with these arguments, Hall, Sora, et al. (2014) found that the serotonin 1B antagonist SB 224289 reduced locomotor activity in DAT

KO mice. However, the effects of serotonin 1B antagonists have not been more widely explored in the DAT KO model. Here, the effects of SB 224289 (and for comparison atomoxetine) on DAT KO-induced hyperactivity and CAR impairments are explored

DAT -/- and DAT +/- mice, as well as in male and female mice.

194

2.0 Materials and methods

2.1 Subjects

Male and female, DAT +/+ and DAT +/- mice were used in these experiments, divided between two treatments groups: atomoxetine (9 DAT +/+, 17 DAT +/-, and 11

DAT -/- female mice; and 8 DAT +/+, 12 DAT +/-, 6 DAT -/- male mice) and SB 224289

(9 DAT +/+, 13 DAT +/-, and 7 DAT -/- female mice and 10 DAT +/+, 11 DAT +/-, 11

DAT -/- male mice). These mice were from the DAT KO line originally developed by

Ichiro Sora (Sora et al., 1998) used to establish a colony at the University of Toledo in

2015. Mice were produced by heterozygous DAT KO crosses. Mice were housed in a standard light cycle 12/12 hrs (lights on at 7am), standard temperature (20-22 °C), and standard humidity (40-60%). At weaning, tissue samples were collected for genotyping by taking ear punches, and then ear tags attached for identification. Mice were genotyped by PCR for DAT genotypes as described in a previous publication (Perona et al., 2008).

All mice were between 2 and 6 months of age. As there is often a greater morbidity in

DAT -/- mice, often in the neighborhood of 50% by adulthood, substantial efforts were taken to improve their survival. Beginning at 4 days postnatal, food pellets were soaked in water for at least one hour and provided to all neonatal mice in the home cage. This procedure was found to substantially increase the survival rate of DAT -/- mice, consistent with some other authors (Del'Guidice et al., 2014). Findings in humans with genetic DAT deficiency include description of an eating dysfunction that may result from difficulty swallowing (Ng et al., 2014). The procedure was continued throughout the experiments. All experiments were conducted in accordance with all applicable

195

guidelines for animal research, including those of the Association for Assessment and

Accreditation of Laboratory Animal Care and the National Institutes of Health (NIH,

USA) under protocols approved by the University of Toledo Institutional Animal Care and Use Committee.

2.2 Locomotor activity

Locomotion was assessed in four white opaque Plexiglas boxes (50×50×40 cm) over 3 hours using an Anymaze video tracking system (Stoelting, Wood Dale, IL). Mice were placed gently in the middle of the testing chamber individually under dim red light in a quiet room. The boxes were cleaned with acetic acid solution between tests. The dependent measure determined using the Anymaze system was distance (m). Mice were tested for locomotor activity 4 times after injections that were given in a pseudo- randomized manner. Separate groups of subjects were tested after injection with different doses of atomoxetine or SB 224289 (see Section 2.4). 5 minutes after injection mice were gently placed in the test boxes. Locomotion was assessed for 60 min after injection. The locomotor activity data (in m) is presented in 5 min time bins.

2.3 Cliff Avoidance Reaction (CAR)

The CAR apparatus consists of a circular platform with a diameter of 20 cm, elevated 50 cm above the floor. DAT +/+ mice normally avoid approaching the edge of the platform, and if they do, they are very cautious, rarely falling. On the other hand,

DAT -/- mice approach the edge in a dangerous manner, even sometimes trying to climb underneath the platform, so that over 50% of them fall from the platform (Yamashita et 196

al., 2013). In this test, mice are placed individually on the platform for 60 min. Their behavior was recorded digitally and subsequently evaluated by an observer blind to genotype. Separate groups of mice were tested after injection with different doses of atomoxetine or SB 224289 (see Section 2.4). Mice were gently placed in the center of the platform 5 min after injection. The duration of the test was 60 min. The platform was cleaned by spring antiseptic Rescue solution between each test. Falling from the platform was considered an impairment in CAR performance, the primary outcome measure being the percentage of each group that were able to remain on the platform for the entire testing period. CAR performance was defined a %CAR = the number of intact CAR mice

(which did not fall from the platform)/ total number of mice tested mice) × 100. The latency to the first fall was also recorded. Mice that fell from the platform were returned to the platform and the number of falls also recorded.

2.4 Drug Treatments

Treatments were administered in counterbalanced fashion (defined by a pseudo-

Williams’ Latin square). Separate groups of mice were tested with atomoxetine and SB

224289. Each dose was at least 4 days apart for washout. The mice were tested 5 minutes after drug administration. Atomoxetine (ATX; (R)-N-Methyl-γ-(2-methyl- phenoxy)benzenepropanamine hydrochloride; Sigma Aldrich, St. Louis, MO) was dissolved in sterile saline (0.9% w/v NaCl) in doses of 0, 1, 2 and 10 mg/kg, IP, in a volume of 10 ml/kg. SB 224289 (1'-methyl-5-[[2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3- yl)biphenyl-4-yl]carbonyl]-2,3,6,7-tetrahydro-spiro[furo[2,3-f]indole-3,4'-piperidine];

Santa Cruz Biotechnology) was dissolved in 10% β hydroxypropyl cyclodextrin in 197

distilled water, and administered in doses of in doses of 0, 10, and 20 mg/kg IP, in a volume of 10 ml/kg. Mice in each group were tested for both locomotion and CAR, but between testing for locomotion and CAR there was a 7-day washout period.

2.5 Statistics and data analysis

The data were analyzed using Graphpad Prism v. 22. Data for each sex were analyzed separately. Multifactorial repeated-measures analysis of variance (ANOVA) was used to analyze the effects of the drugs on locomotor activity separately for each

DAT genotype group (DAT +/+, DAT +/- and DAT -/-), and separately for each sex using the within-subjects factors of treatment Dose (ATX: 0, 1, 2, and 10 mg/kg groups;

SB 244289: 0, 10, and 20 mg/kg groups) and Time, divided into 5 min time bins. If a significant Mauchly's Test of Sphericity result was obtained the results were adjusted using a Greenhouse–Geisser correction of degrees of freedom to provide more conservative significance estimates. Whenever applicable, significant ANOVA results were further analyzed by Bonferroni post hoc means comparisons. Data are presented as mean ± SEM. Statistical significance was set at p < 0.05.

CAR impairment effects (% CAR) were evaluated using the Chi square test, and the data is expressed as a percentage. Statistical significance was set at p < 0.05.

Continuous variables in the CAR dataset, stretch attend postures, head dips, fecal boli, and falls, as well as the latency to fall, were evaluated by ANOVA as described above for locomotion. Data are presented as mean ± SEM. Statistical significance was set at p <

0.05.

198

3.0 Results

3.1 Effects of Atomoxetine on locomotor activity in DAT +/+, DAT +/-, and DAT -/- mice

The effects of ATX on Figure 6 locomotor activity in male DAT +/+,

DAT +/- and DAT -/- mice are represented in Fig. 1. Locomotion in

DAT -/- mice did not habituate over time, as was observed for DAT +/+ and DAT +/- mice. This was confirmed by a significant main effect of Genotype in an ANOVA that examined the vehicle data alone

(F (2, 31) = 135.4, p < 0.001). This main effect in the ANOVA was confirmed by post hoc means comparisons that found that DAT-/- male mice had significantly elevated locomotor activity in comparison with both DAT +/+ and DAT +/- male mice at all timepoints after saline administration (p < 0.001). Figure 1. Locomotor activity in male (A) DAT +/+, (B) DAT +/-, and (C) DAT -/- mice after ATX did significantly affect administration of saline or 1, 2, or 10 mg/kg ATX. *** significant post hoc comparisons vs. saline. locomotor activity in DAT+/+ or 199

DAT -/- male mice (F (1.6, 9.8) = 3.3, Figure 2 NS and (F (1.4, 5.5) = 2.2, NS), respectively; Figs. 1A and 1C). There was a strong trend toward a reduction in locomotor activity in male DAT

+/+ mice at the highest dose.

However, ATX caused a significant reduction in locomotor activity in male DAT +/- mice (F (2.6, 28.6) =

12.3, p < 0.0001; Fig. 1B).

The effects of ATX on locomotor activity in female DAT +/+, DAT +/- and DAT -/- mice are pictured in Fig.

2. Locomotion in DAT -/- mice did not habituate over time, as was observed for DAT +/+ and DAT +/- mice. Female DAT -/- mice were profoundly hyperactive compared to female DAT +/+ mice, but female

DAT +/- mice were not more active than female DAT +/+ mice. This was Figure 7. Locomotor activity in female (A) DAT +/+, (B) DAT +/-, and (C) DAT -/- mice after confirmed by a significant effect of administration of saline or 1, 2, or 10 mg/kg ATX. *** significant post hoc comparisons vs. saline. Genotype in an ANOVA for the 200

vehicle data alone (F (2, 20) = 78.6, p < 0.001), and confirmed by post hoc means comparisons showing that locomotor activity was significantly higher in DAT -/- female mice than in female DAT+/+ or DAT+/- mice. ATX produced significant reductions in locomotor activity in female DAT +/+ mice (F (2, 31) = 135.4, p < 0.0001; Fig. 2A), which was confirmed by post hoc means comparisons that revealed significant reductions in locomotor activity after both 2 and 10 mg/kg ATX (p < 0.05 vs. saline). Locomotion was reduced throughout the test session, and significant reductions were observed for the first half of the testing period. ATX also significantly reduced locomotor activity in female DAT +/- mice (F (4.2, 229.8) = 7.1, p < 0.05; Fig. 2B), which also was confirmed by post hoc means comparisons that confirmed that 10 mg/kg ATX reduced locomotion

(p < 0.05 vs. saline). Significant differences were observed only at the first few time- points. Locomotor was much higher in female DAT -/- mice, and there was not habituation over the test period. ATX slightly reduced locomotion, which was confirmed by a significant main effect of ATX Dose in the ANOVA (F (2.1, 14.5) = 4.1, p < 0.05;

Fig. 2C), and was confirmed by post hoc means comparisons showing that 10 mg/kg

ATX reduced locomotor activity in the first 5 time-bins (p < 0.05).

3.2 Effects of SB 224289 of locomotor activity in DAT +/+, DAT +/-, and DAT -/- mice

The effects of SB 224289 on locomotor activity in male DAT +/+, DAT +/- and

DAT -/- mice are pictured in Fig. 3. Male DAT -/- mice were profoundly hyperactive compared to male DAT +/+ mice, but male DAT +/- mice were not more active than male DAT +/+ mice. This was confirmed by a significant main effect of Genotype in an

ANOVA for the vehicle data alone (F (2, 31) = 135.4, p < 0.001). As can be seen in Fig. 201

3A, there was no effect of SB Figure 3 224289 on locomotion in DAT +/+ male mice either for the main effect of SB224289 dose (F (2.4, 21.4) =

2.8, NS) or the interaction between

SB224289 Dose and Time (F (5.2,

46.9) = 1.6, NS). In contrast, DAT

+/- male mice showed a reduction in locomotor activity after injection with SB 224289 over much of the session, which was shown by a significant effect of SB 224289

Dose in the ANOVA (F (2.2, 28.0)

= 4.7, p < 0.05; Fig. 3B). These effects were confirmed by post hoc comparisons that revealed significant reductions in locomotor activity after treatment with 10 and

20 mg/kg SB224289 between 15 and 30 minutes of the test. SB Figure 3. Locomotor activity in male (A) DAT 224289 also reduced locomotor +/+, (B) DAT +/-, and (C) DAT -/- mice after administration of saline or 10, 20 or 30 mg/kg activity in male DAT -/- mice, SB 224289. * significant post hoc comparisons vs. saline. which was shown by a significant 202

main effect of SB 224289 Dose in the ANOVA (F (2.5, 22.8) = 4.4, p < 0.05; Fig. 3C).

Figure 4 This was confirmed by post hoc

comparisons showing that

locomotion was reduced at

doses of 10 and 20 mg/kg SB

224289 at all time-points after

15 min (p < 0.05, and p < 0.01

respectively). It should be noted

that locomotion in DAT -/- mice

did not habituate over time, as

was observed for DAT +/+ and

DAT +/- mice. The effect of SB

224289 did not restore this

habituation, but rather had

effects across the entire test

session, although these were

only significant later in the test

session.

locomotor activity in female

DAT +/+, DAT +/- and DAT -/-

Figure 4. Locomotor activity in female (A) mice are pictured in Fig. 4. DAT +/+, (B) DAT +/-, and (C) DAT -/- mice after administration of saline or 10, Female DAT -/- mice were 20 or 30 mg/kg SB 224289. * significant post hoc comparisons vs. solvent. profoundly hyperactive 203

compared to female DAT +/+ mice, while female DAT +/- Figure 5 mice were slightly more active than female DAT +/+ mice and did not show habituation of locomotion. These genotypic differences were confirmed by a significant main effect of Genotype in an ANOVA comparing the vehicle data alone (F (2, 26) = 25.4, p < 0.0001). SB 224289 did not affect locomotor activity in female DAT +/+ mice (F (1.7,

11.7) = 0.5, NS; Fig. 4A), female DAT +/- mice (F (2.1,

16.5) = 1.3, NS; Fig. 4B), or female DAT -/- mice (F (1, 16)

= 1.1, NS, Fig. 4C).

3.3 Cliff Avoidance Reaction (CAR) deficits in DAT KO mice

Fig. 5 shows pairwise comparisons for successful CAR performance as a factor of sex in each DAT genotype under baseline conditions (no injections). Male mice consistently showed reduced CAR performance compared to female mice. In DAT +/+ and DAT +/- mice these sex differences were small in magnitude, although statistically significant.

Female DAT +/+ mice had significantly higher CAR performance than male DAT +/+ mice (χ2(1) = 5.4, p < 0.05;

Fig. 5, top). Female DAT +/- mice also showed significantly

2 higher CAR performance than male DAT +/- mice (χ (1) = Fig. 5 Effects of sex on CAR. *P < 0.05; P < 5.2, p < 0.05; Fig. 5, middle). Much larger effects were 0.0001 204

observed in female DAT -/- mice compared to male DAT -/- mice (χ2(1) = 40.6, p <

0.0001; bottom). Indeed, the majority of male DAT -/- fell from the platform, whereas less than half of the female Figure 6

DAT -/- mice fell from the platform.

In the CAR test, male and female DAT +/+ mice exhibited successful

CAR performance. Their behavior in the task was characterized by very cautious approaches to the edge of the small, circular platform. Although the apparatus is small, this does involve and approach- avoidance conflict. The animals are certainly anxious on the exposed platform, and cautious about Figure 6. Genotypic comparisons between male and approaching the edge, but at female, DAT +/+, DAT +/-, and DAT -/- mice in the CAR test. * P < 0.05; ***P < 0.001; ****P < the same time desire to 0.0001. extricate themselves from the platform entirely, which promotes exploration of the edge 205

in a search for an escape route. In DAT +/+ mice this is minimal, and the primary behavior is characterized by few approaches to the edge, maintained position in the center of the platform, and a flat body posture in which the mouse almost seems to be pushing itself into the platform. The initial exploration of Figure 7 the edge of the platform usually lasts for 5 to 10 mins and then DAT +/+ mice largely settle in the middle of the platform, with occasional exploratory bouts at about 5 min intervals. By contrast, both male and female DAT -/- mice exhibit much more continuous, extensive and incautious exploration of the edge of the platform, characterized by numerous head dip behaviors, in which the upper torso of the animal is placed over the edge of the platform, and the head dips below the level of the bottom of the platform. This often places the center of the animal’s weight perilously close to the edge, resulting in falls. It is also interesting to note that the foot position of the DAT +/+ and DAT -/- mice is quite different. DAT +/+ keep their feet Figure 7. Effects of atomoxetine in away from the edge of the platform, whereas male DAT +/+, DAT +/-, and DAT -/- mice in the CAR test. * P < 0.05; DAT -/- often reach over the edge of the **P < 0.01; ***P < 0.001. platform, placing their feet on the side of the 206

platform, which also acts to shift their center of gravity Figure 8 perilously close to the edge of the platform. This behavior is consistent with that reported by Yamashita et al. (2013).

Figure 6 shows pair-wise genotypic comparisons of successful performance in the CAR test under baseline conditions (no injections). CAR performance was slightly, but significantly, impaired in male DAT +/- mice compared to male DAT +/+ (χ2(1)

= 5.6 p < 0.05; Fig. 6A). There was no different in

CAR performance between female DAT +/+ and female DAT +/- mice (Fig. 6B). Male DAT -/- mice had impaired CAR performance compared to male

DAT +/+ mice (χ2(1) = 69.6, p < 0.0001; Fig. 6C).

Female DAT -/- mice also had impaired CAR performance compared to female DAT +/+ mice (χ2(1)

= 58.7, p < 0.0001; Fig. 6D), although the magnitude of this effect was much smaller than the effect observed in male mice. Male DAT -/- mice were impaired compared to male DAT +/- mice as well Figure 8. Effects of atomoxetine in female DAT +/+, DAT +/-, (χ2(1) = 20.3, p < 0.0001; Fig. 6E), and female DAT -/- and DAT -/- mice in the CAR test.* P < 0.05; **P < 0.01; mice were also impaired compared to female DAT +/- ***P < 0.001 mice (χ2(1) = 14.0, p < 0.001; Fig. 6F). 207

3.4 Effects of Atomoxetine (ATX) on CAR in DAT KO mice

The effects of ATX on CAR performance was Figure 9 different in male mice of different DAT genotypes

(Fig. 7). Car performance in male DAT +/+ mice was significantly impaired by ATX in a dose-dependent manner (Fig. 7A). Significant reductions in successful

CAR (CAR%) were observed after ATX treatment

(χ2(3) = 21.5, p < 0.001). These effects were significant for the 2 and 10 mg/kg doses of ATX (p < 0.001). In contrast to these impairments in CAR performance observed in male DAT +/+ mice, male DAT +/- and male DAT -/- mice showed substantial improvements in CAR performance after ATX administration. The initially-impaired CAR performance of male DAT +/- mice was significantly improved by ATX treatment in a dose-dependent manner (χ2(3) = 29.7, p < 0.001; Fig.

7B). CAR performance was significantly improved at the 2 and 10 mg/kg doses of ATX (p < 0.05) and p <

0.01, respectively). The initial CAR performance of male DAT -/- mice was much more impaired than it Figure 9. Effects of SB was in male DAT +/- mice (Fig. 7C compared to Fig 224289 in male DAT +/+, DAT +/-, and DAT -/- mice 7B). CAR performance was significantly improved by in the CAR test. * P < 0.05; ***P < 0.001 ATX in DAT -/- mice in a dose-dependent manner 208

(χ2(3) = 196.0, p < 0.001). These effects were significant for the 2 and 10 mg/kg doses of

ATX (p < 0.05 and p < 0.001, respectively).

In male DAT -/- mice CAR impairments were reduced in Figure 10 a dose-dependent manner by ATX (χ2 (3) = 196.0, p < 0.001; see

Fig. 5B). Intriguingly, in male DAT +/- mice CAR performance was improved in dose-dependent manner by ATX at 2 and 10 mg/kg, but not 1 mg/kg ATX (χ2(3) = 27.7; p < 0.05 and p <

0.001, respectively). Performance of male DAT +/+ mice in

CAR deteriorated after injection with 2 or 10 mg/kg ATX (χ2 (3)

= 21.5, p < 0.001).

The effect of ATX on CAR performance in female mice is shown in Fig. 8. Unlike what was observed in male DAT +/+ mice, CAR performance was unaffected by ATX in female DAT

+/+ mice (Fig. 8A; χ2(3) = 6.9, NS). Performance after ATX treatment was slightly enhanced compared saline treatment, but the performance of saline-treated female mice was already near maximal levels. The basal levels of performance in female DAT

+/- mice (Fig. 8B) was only slightly impaired, but ATX did improve CAR performance in these mice (χ2(3) = 20.1, p = Figure 10. Effects of SB 224289 in female 0.01). This effect in DAT +/- mice was significant only for the DAT +/+, DAT +/-, and DAT -/- mice in highest dose of ATX (p < 0.0001). CAR performance was the CAR test. ***P < 0.001 substantially impaired in female DAT -/- mice (Fig. 8C). ATX completely reverse this deficit (χ2(3) = 107.8, p < 0.001), and this effect was statistically 209

significant for the 10 mg/kg dose of ATX (p < 0.0001).

3.5 Effects of SB 224289 on CAR in DAT KO mice

Effects of SB 224289 treatment on CAR performance in male mice of different

DAT genotypes is shown in Fig. 9. In male DAT +/+ mice CAR performance was impaired by SB 224289 (χ2(3) = 28.6, p < 0.0001; Fig. 9A). This effect of SB 224289 in male DAT +/+ mice was significant at the 30 mg/kg dose of SB 224289 In male DAT +/- mice, the moderately-impaired baseline CAR performance was slightly, but significantly, improved by SB224289 (χ2(3) = 8.3, p < 0.05). This slight improvement was significant at the 20 mg/kg dose of SB 224289 (p < 0.05), but not at the 30 mg/kg dose. In male

DAT -/- mice CAR performance was also slightly, but significantly, improved by treatment with SB 224289 (χ2(3) = 196.0, p < 0.05). This slight improvement was only significant at the 30 mg/kg dose of SB224289 (p < 0.05). This pattern of responses of SB

224289 on CAR performance was similar to that observed for ATX, but the effects were much smaller and not as clearly dose-dependent.

Effects of SB 224289 treatment on CAR performance in female mice are shown in Fig. 10. No effects of SB 224289 were observed in either female DAT +/+ mice (Fig.

10A) or female DAT +/- mice (Fig. 10B). Performance in both groups was high initially, although there was still room for improvement as is clear from the effects of ATX in a previous experiment (Fig. 8B). Female DAT -/- mice had substantially impaired CAR performance that was significantly improved by SB 224289 (χ2(3) = 35.5, p < 0.001; Fig.

10C), although this was significant only at the 10 and 20 mg/kg doses of SB 224289 (p <

0.001) and not at the highest dose. 210

4.0 Discussion

In the present study, male and female DAT -/- mice were hyperactive and showed impairments in CAR testing. The CAR deficits are likely to reflect some type of impulsivity or impaired decision-making or risk assessments. These effects confirm previous reports of hyperactivity (Giros et al., 1996; Sora et al., 1998) and CAR impairments (Yamashita et al., 2013) in DAT -/- mice. A slight hyperactivity was also observed in DAT +/- mice in some of the present experiments for males (e.g. Fig. 1, but not Fig. 2), but not females, although there was a trend toward hyperactivity in females in one experiment (Fig. 4). It thus appears that male mice may show a greater propensity towards hyperactivity after partial DAT deletion. CAR deficits were also observed in

DAT +/- mice under some conditions, although they were less robust than the deficits observed in DAT -/- mice. These behavioral deficits provide face validity for both DAT -

/- and DAT +/- as models of ADHD-like deficits, perhaps representing a range from mild

(DAT +/-) to more sever (DAT -/-) ADHD-like deficits. If this depiction is valid it suggests that the more severe condition may also be associated with more co-morbidities, as has been observed in humans with dopamine transporter deficiency syndrome (Ng et al., 2014), and perhaps the broader co-morbidities that characterize ADHD (Biederman,

Newcorn, & Sprich, 1991). A similar pattern of effects has been suggested for SERT -/- and SERT +/- mice as an animal model of Autism Spectrum Disorder (Tanaka et al.,

2018).

The present results provide additional evidence for the face and predictive validity of both the DAT -/- and DAT +/- models of ADHD. The validity of the DAT -/- model is well-established (Arime et al., 2011; de la Pena et al., 2018), although the validity of the 211

DAT +/- is still uncertain. This uncertainty comes from this model being somewhat less- studied than the DAT -/- model. In DAT +/- mice the expression of DAT protein is about

50% of what is seen in DAT +/+ mice, tissue dopamine content is slightly reduced, dopamine D1 and D2 receptor expression is reduced, and extracellular dopamine levels are about two times higher (Giros et al., 1996; Jones et al., 1998). These changes are quite modest compared to DAT -/- mice. The behavioral phenotypes typically observed in DAT -/- mice are also milder or absent in DAT +/- mice. Locomotor activity is either absent (Giros et al., 1996; Sora et al., 1998), or mild (Hall, Itokawa, et al., 2014; Mereu et al., 2017; Spielewoy et al., 2000) in DAT +/- mice. Impairments in PPI (Mereu et al.,

2017; Ralph et al., 2001), and the marble burying task (Fox et al., 2013) are not seen in

DAT +/- mice, but some other changes, such as immobility in the tail suspension test, are observed equally in DAT -/- mice and DAT +/- mice (Perona et al., 2008). The present experiments are the first to show that DAT +/- mice have impairments in the CAR test.

The nature of these effects is a matter of some debate (see discussion in Yamashita et al.

(2013)), but may either reflect impulsivity or some aspect of executive function or risk assessment. DAT +/- mice do show impairments in a number of cognitive tests, including novel object recognition (Franca et al., 2016; Mereu et al., 2017), the reversal stage of an attentional set-shifting task (Cybulska-Klosowicz, Dabrowska, Niedzielec, Zakrzewska,

& Rozycka, 2017; Cybulska-Klosowicz, Laczkowska, Zakrzewska, & Kaliszewska,

2017), and in measures of attention and impulsivity in the 5-Choice serial reaction time task (5-CSRTT) (Mereu et al., 2017). Although the deficits in the CAR test could be considered to reflect some type of impulsive deficit, it is likely that this may reflect some deficit in decision making or some other aspect of executive function in addition. Indeed, 212

children with ADHD make many inherently risky choices (Dekkers et al., 2018), that are quite concerning to their parents and caretakers, symptoms which are not generally improve by current treatment approaches. There is some evidence for impulsivity in DAT

+/+, for instance from the 5-Choice Serial Reaction Time Task (Mereu et al., 2017). It will be important to better specify these deficits using other cognitive tests in DAT +/- mice, and also to specifically target these symptoms for the treatment of ADHD.

Many of the behavioral deficits in DAT -/- mice can be improved by effective ADHD treatments (Dekkers et al., 2018; Gainetdinov et al., 1999; Uchiumi et al., 2013;

Yamashita et al., 2006; Yamashita et al., 2013). Del'Guidice et al. (2014) found that ATX enhanced cognition flexibility in DAT -/- but did not ameliorate locomotor hyperactivity.

The present results also found that ATX did not reduce hyperactivity in male DAT -/- mice, but was effective in female DAT -/- mice. That study did use a limited dose-range as well, and the present study did show that higher doses were effective. The present experiments extend previous findings demonstrating predictive validity of both the DAT

-/- and DAT +/- models, but also raises certain cautions. In some cases, effects are not always observed in DAT +/- mice, and the effects are somewhat sex-dependent, and not being observed in both sexes, and sometimes producing impairments in one sex for certain genotypes. This may also provide a model to study sex differences in ADHD that might affect treatment.

Although not well explored, there is some evidence that ADHD-like impairments can be improved in DAT +/- mice by effective ADHD treatments. Amphetamine treatment for five days normalized DAT +/- performance in a modified version of the 5-

CSRTT (Mereu et al., 2017), although it is not clear from that study whether 213

improvements might have been due to continued testing due to lack of saline controls during this period of testing. That study also showed reductions in hyperactivity after amphetamine treatment in DAT +/- mice. The present results showed that both hyperactivity and CAR deficits could be improved by ATX, adding to evidence for the predictive validity of the model.

If the DAT KO model does have the ability to identify effective ADHD treatments, then it should also be possible to identify novel treatments using the model. In a previous study we found that the 5-HT1B antagonist SB 224289 reduced hyperlocomotion in DAT -/- mice (Hall, Sora, et al., 2014). The present study confirmed those findings; indeed, SB 224289 reduced locomotor hyperactivity in both male DAT -/- and DAT +/- mice. However, it was without effect on locomotor activity in female mice of any genotype. Additionally, in the CAR test SB 224289 had some effects on impairments in male DAT -/- and DAT +/- mice, as well as in female DAT -/- mice, although these effects were modest and lacked the strong dose-dependency seen for ATX in this test. This may perhaps be because different 5-HT1B receptor populations have different effects on behavior in this test. Indeed, SB 224289 impaired CAR in both male and female DAT +/+ mice, and female DAT -/- mice had an inverted U-shaped response to SB 224289, with effects only observed at the lower two doses. Nonetheless, this data does provide additional evidence that SB 224289 antagonism might be an effective treatment approach for ADHD-like behavioral deficits, at least enough to warrant further inquiry.

There are a number of reasons to think that 5-HT1B antagonism might be useful in the treatment of cognitive dysfunctions in ADHD. Serotonin 1B antagonism increases 214

the release of acetylcholine in the prefrontal cortex (Hu et al., 2007), but serotonin 1B agonists have been reported to have the same effects (Consolo et al., 1996). Reduced serotonin 1B receptor expression in serotonin 1B receptor knockout mice reduces acetylcholine levels in prefrontal cortex resulting in some cognitive impairments

(Consolo et al., 1996; Wolff, Benhassine, et al., 2003). However, these sorts of constitutive knockouts might produce impairments in different serotonin 1B receptor populations that have differential effects on behavioral responses. This was suggested to explain differences between the effects of partial and complete depletions of 5HT1B receptors in knockout mice (Hall, Sora, et al., 2014). For instance, 5HT1B receptors also modulate dopamine release in the striatum (Sarhan, Cloez-Tayarani, Massot, Fillion, &

Fillion, 1999) in addition to their effects in the prefrontal cortex. Glutamate release in the hippocampus is also modulated by serotonin 1B receptors (Wolff, Savova, et al., 2003).

Despite this complexity of serotonin 1B receptor effects, the serotonin 1B receptor antagonist SB 224289 has been found to facilitate learning and to reverse memory and learning impairments induced by scopolamine (Gaster et al., 1998; Selkirk et al., 1998).

Serotonin 1B knockout mice also have enhanced spatial memory (Meneses, 1999). SB

224289 also reverses consolidation deficits produced by scopolamine or dizocilpine

(Meneses, 2001). Serotonin depletion attenuates SB 224289 facilitation of memory consolidation suggesting that the effects depend on serotonin release (Meneses, 2001).

The present results add to these studies, indicated that SB 224289 can also reverse

ADHD-like deficits, including those in the CAR test. It will be interesting to determine if

DAT KO-induced deficits in other tests of cognition and attention can also be ameliorated by SB 224289. 215

In conclusion, the present study confirmed that DAT -/- mice are hyperactive and have deficits in CAR test. In both cases these deficits were ameliorated by an effective

ADHD treatment, at to some extent by a putative treatment, 5HT1B receptor antagonism.

This study extended previous findings by demonstrating that DAT +/- mice also show similar, if less robust deficits, that are also ameliorated by both ATX and the 5HT1B receptor antagonist SB 224289. These results support the view that DAT KO mice, including DAT +/- mice, model aspects of ADHD, and that these models can be used to evaluate novel ADHD therapeutics. Additional work, examining other ADHD-like deficits in more complex tests of attention and cognition, and the potential for 5-HT1B antagonism to reverse those deficits, are encouraged by the present results.

216

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knockout mice. PLoS One, 7(12), e51455. doi:10.1371/journal.pone.0051455

Wong, P., Sze, Y., Chang, C. C., Lee, J., & Zhang, X. (2015). Pregnenolone sulfate

normalizes schizophrenia-like behaviors in dopamine transporter knockout mice

through the AKT/GSK3beta pathway. Transl Psychiatry, 5, e528.

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Sora, I. (2006). Norepinephrine transporter blockade can normalize the prepulse

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doi:10.1007/s00213-013-3009-9

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Chapter 6

Heterozygous dopamine transporter knockout mice as an animal model of ADHD: effects of amphetamine and the serotonin 1B receptor antagonist SB224289

a, b a Yasir H. Saber , and F. Scott Hall , a Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, OH, USA; b Ninevah College of medicine, Ninevah university, Mosul, Iraq

Manuscript in Preparation for submission to Neuropsychopharmacology.

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Abstract

Introduction: Dopamine is a neurotransmitter that plays a major role in attention, working memory, reward, motivation, and motor activity. Perturbations in dopamine signaling have been implicated in several developmental psychological disorders, including attention deficit/ hyperactivity disorder (ADHD), schizophrenia, and obsessive- compulsive disorder. The dopamine transporter (DAT) is a critical regulator of dopamine release dynamics. Developmentally, dopamine activity influences prefrontal cortex maturation, with consequent effects on response inhibition ability and decision making. It has been suggested that DAT KO mice may model aspects of ADHD. DAT +/- mice do not show many behavioral effects observed in homozygous DAT KO (DAT -/-) mice, but they have not been well-studied in tests of attention and cognitive function. Therefore,

DAT +/- mice were examined in the 5-choice continuous performance (5-CCPT) task to assess attention and impulsivity, as well as the effects of amphetamine and the 5-HT1B antagonist SB 224289.

Methods: Male DAT +/- and DAT wildtype (DAT +/+) mice were examined in the 5-

CCPT. A modified short version of 5-CCPT was used. After stable performance was attained mice were tested in challenge sessions after being injected with different doses of amphetamine (0, 0.3, 0.66, and 1.5 mg/kg IP). After a 10-day wash-out period with continued baseline testing, mice were tested with the serotonin 1B receptor antagonist SB

224289 (0, 10, 20 mg/kg IP).

Results: In the 5-CCPT DAT +/- mice made more premature errors and had a higher percentage of incorrect responses that DAT +/+ mice. These impairments were reduced by amphetamine administration. The high dose of amphetamine also increased the 230

sensitivity index in DAT +/- mice. DAT +/- mice also showed fewer premature errors after administration of SB 224289.

Conclusions: In the present study, DAT +/- mice showed evidence of motor impulsivity and impaired attention in the 5-CCPT. Amphetamine reduced this impulsivity in a dose dependent manner. One of the current goals of preclinical ADHD research is to identify non-stimulant approaches to the treatment of ADHD. The 5-HT1B antagonist SB 224289 also reduced impulsivity in the 5-CCPT. This data suggests that DAT +/- mice may model key aspects of ADHD and may provide a useful approach to identifying new treatments for ADHD.

Keywords: Dopamine transporter, cognition, impulsivity, attention, atomoxetine, serotonin 1B receptor, SB 224289, ADHD, 5-choice continuous performance test

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1.0 Introduction

Attention deficit/hyperactivity disorder (ADHD) is one of the most prevalent developmental neuropsychological disorders in children. ADHD has been reported to affect between 5.3 and 11% of the school-age children (Lecendreux, Konofal, Cortese, &

Faraone, 2015; Polanczyk, Willcutt, Salum, Kieling, & Rohde, 2014; Visser et al., 2014) and 3.4% of the adult population (Fayyad et al., 2007). ADHD incidence has continued to increase over the last decade, especially among adults and females (Fairman, Peckham, &

Sclar, 2017), which may reflect more accurate diagnosis within these groups, but may also reflect actual increases in ADHD incidence. ADHD has a negative impact on academic and professional achievement, social relationships, and aspects of daily living, and consequently produces a substantial emotional and financial burden for those affected, their families and communities (de Zeeuw, van Beijsterveldt, Ehli, de Geus, &

Boomsma, 2017; Fenesy, Teh, & Lee, 2019; Rietveld & Patel, 2019; Zendarski, Mensah,

Hiscock, & Sciberras, 2019; Zendarski, Sciberras, Mensah, & Hiscock, 2017).

There is genetic evidence for involvement of variation in the gene for DAT

(SLC6A3) in behavioral phenotypes associated with ADHD and with ADHD itself.

ADHD is highly heritable (Genro et al., 2008; Grunblatt, Werling, Roth, Romanos, &

Walitza, 2019; Hong, Hwang, Lim, Kwon, & Jin, 2018; Yang et al., 2007), but the genetic underpinnings of ADHD are highly polymorphic and heterogenous, with each gene variant making only a small individual contribution (Faraone & Larsson, 2019).

DAT gene variants are not identified in genome-wide association studies (GWAS)

(Demontis et al., 2019), perhaps for those reasons. A number of different, rare mutations in DAT are associated with ADHD (Hansen et al., 2014; Mergy et al., 2014), consistent 232

both with a role of DAT in ADHD and with the view that the genetic variants underlying this relationship may be individually rare but numerous. Imaging studies ADHD patients also support a role of alterations in DAT function, sometimes reductions (Hesse,

Ballaschke, Barthel, & Sabri, 2009), but more often increased DAT function (Cheon et al., 2003; Krause, Dresel, Krause, la Fougere, & Ackenheil, 2003; Spencer et al., 2007).

DAT gene polymorphisms have also been associated more broadly with functions that may underlie ADHD, including time perception that can affect learning in reward tasks, executive function and cognition (Marinho et al., 2018). Parkinsonian patients show reductions in DAT expression in the striatum, as a compensatory response to loss of dopamine, and these patients also have deficits in mental timing that are correlated with reduced DAT expression (Avanzino et al., 2016; Breska & Ivry, 2018; Honma, Kuroda,

Futamura, Shiromaru, & Kawamura, 2016).

Although the clinical data does not consistently support the idea that reductions in

DAT expression underlie ADHD, studies in genetically modified mice have shown that reductions in DAT expression produce ADHD-like phenotypes that are reversed by drugs that treat ADHD. Homozygous DAT knockout (DAT -/-) mice exhibit locomotor hyperactivity (Giros, Jaber, Jones, Wightman, & Caron, 1996; Sora et al., 2001; Sora et al., 1998). This activity is highly repetitive, invariant, and perseverative (Fox, Panessiti,

Hall, Uhl, & Murphy, 2013; Ralph, Paulus, Fumagalli, Caron, & Geyer, 2001). DAT -/- mice also have deficits in prepulse inhibition of acoustic startle (PPI) (Arime, Kasahara,

Hall, Uhl, & Sora, 2012; Ralph et al., 2001; Wong et al., 2012; Wong, Sze, Chang, Lee,

& Zhang, 2015; Yamashita et al., 2006; Yamashita et al., 2013). PPI is not a test of attention per se but has been taken to represent the presence of such deficits. DAT -/- 233

mice have also been shown to have impairments in the cliff avoidance reaction

(Yamashita et al., 2013), which are thought to reflect deficits in impulse control or executive function. DAT -/- mice also have deficits in a variety of learning tasks

(Gainetdinov et al., 1999; Li, Arime, Hall, Uhl, & Sora, 2010; Morice et al., 2007; Wong et al., 2012; Wong et al., 2015). It is likely that many of these deficits reflect indirect effects of other behavioral phenotypes, such as hyperactivity, attentional impairments, or impulsivity, on learning, just as is seen in ADHD.

ADHD-like phenotypes observed in DAT -/- mice can be reversed by drugs used to treat ADHD like amphetamine and methylphenidate, including hyperactivity

(Gainetdinov et al., 1999), PPI deficits (Yamashita et al., 2006), and the cliff avoidance reaction test (Yamashita et al., 2013). Consistent with the high rate of smoking in ADHD patients (van Amsterdam, van der Velde, Schulte, & van den Brink, 2018), that is likely a form of self-treatment, nicotine also reduces hyperactivity and PPI deficits (Uchiumi et al., 2013) and improves learning deficits (Weiss, Nosten-Bertrand, McIntosh, Giros, &

Martres, 2007; Weiss, Tzavara, et al., 2007) in DAT -/- mice. PPI and CAR deficits in

DAT -/- mice are also reversed by nisoxetine (Yamashita et al., 2006; Yamashita et al.,

2013), a selective NET inhibitor like atomoxetine, and these effects are mediated by actions in the prefrontal cortex (Arime et al., 2012). This and other data suggests that the deficits in DAT -/- mice arise from cellular and circuit level changes in the prefrontal cortex and striatum that involve reduced corticostriatal activity and an imbalance between cortical and subcortical dopamine function (Berlanga et al., 2011; Costa et al., 2006; Cyr et al., 2003; Cyr, Caron, Johnson, & Laakso, 2005; Kasahara, Arime, Hall, Uhl, & Sora,

2015; Shen et al., 2004; Xu et al., 2009; Zhang et al., 2010). This aspect of the DAT KO 234

model fits with the hypothesis that noradrenergic dysfunctions underlie ADHD and are associated with frontostriatal impairments (Biederman & Spencer, 1999).

DAT -/- mice clearly exhibit behavioral phenotypes consistent with all of the major symptoms of ADHD (Diagnostic and statistical manual of mental disorders :

DSM-5, 2013): hyperactivity, impulsivity, impaired attention, and impaired executive function/decision making. However, attempts to assess the behavior of DAT -/- mice in more complex tests of cognitive and attentional function have been unsuccessful (Saber and Hall, unpublished findings). Briefly, DAT -/- mice were found to be incapable of learning even the basic versions of operant tasks necessary to assess attention and other cognitive functions. Moreover, in evaluating DAT -/- mice as a potential animal model of

ADHD it is important to note that complete elimination of DAT function is not observed in ADHD. Heterozygous DAT KO (DAT +/-) mice, with 50% of normal DAT expression might be a better model for ADHD, especially given the extremity of the deficits observed in DAT -/- mice. DAT +/- mice have less extreme alterations in dopamine function that include slightly reduced tissue dopamine content, D1 and D2 dopaminergic receptor expression, and about two times higher extracellular dopamine levels compare with WT mice (Giros et al., 1996; Jones et al., 1998). In contrast to the profound locomotor activity observed in DAT -/- mice, locomotion is only slightly higher in DAT

+/- mice and was not significantly elevated in initial studies (Giros et al., 1996; Sora et al., 1998), but was shown to be slightly elevated in some subsequent studies (Hall,

Itokawa, et al., 2014; Mereu et al., 2017), especially after repeated testing (Spielewoy et al., 2000). Some other deficits found in DAT -/- mice are not observed in DAT +/- mice, including impaired PPI (Mereu et al., 2017; Ralph et al., 2001), and impairments in the 235

marble burying task (Fox et al., 2013).

It is possible that for other ADHD phenotypes that have not been thoroughly investigated, DAT +/- mice might show more modest deficits that may better model the magnitude of deficits observed in ADHD. In tests of cognitive function, impairments in novel object recognition (Franca et al., 2016; Mereu et al., 2017), in the reversal stage of an attentional set-shifting task (Cybulska-Klosowicz, Dabrowska, Niedzielec,

Zakrzewska, & Rozycka, 2017; Cybulska-Klosowicz, Laczkowska, Zakrzewska, &

Kaliszewska, 2017), and in measures of attention and impulsivity in the 5-Choice serial reaction time task (5-CSRTT) (Mereu et al., 2017) have been observed in DAT +/- mice.

In the 5-CSRTT task DAT +/- mice were impulsive and showed some evidence of impaired attention. Amphetamine treatment normalized DAT +/- performance (Mereu et al., 2017), although this may have just resulted from continued testing. Further evaluation of attentional, impulsive, and cognitive deficits in DAT +/- mice are needed, including a wider evaluation of agents effective in the treatment of ADHD.

This model might also be used to evaluate novel non-stimulant approaches to the treatment of ADHD. Hall, Sora, Hen, and Uhl (2014) showed that the serotonin 1B antagonist SB 224289 reduced locomotor activity in DAT KO mice, which might constitute a novel drug target for ADHD. Serotonin 1B receptors are localized on both dendrites/cell bodies and at presynaptic terminals they control release of serotonin or other neurotransmitters (Peddie, Davies, Colyer, Stewart, & Rodriguez, 2008).

Modulation of serotonin 1B receptors might modulate behavior through several mechanisms that might be relevant to the effects of SB 224289 in DAT -/- mice.

Serotonin 1B antagonism increases the release of acetylcholine in the prefrontal cortex 236

(Hu, Wang, Stenfors, Ogren, & Kehr, 2007), which might have consequences on attention and cognition. Serotonin 1B KO mice show greater cognitive flexibility which might be due to increased acetylcholine release in the prefrontal cortex or hippocampus or it enhanced glutamate release in the hippocampus (Wolff et al., 2003). Indeed,

SB224289 facilitates learning consolidation and reverses memory and learning impairments induced by scopolamine (Gaster et al., 1998; Selkirk et al., 1998). Several studies show that genetic polymorphisms in the serotonin 1B receptor gene are associated with ADHD in humans (Guimaraes et al., 2009; Hawi et al., 2002; Ickowicz et al., 2007;

Quist et al., 2003; van Rooij et al., 2015).

The aims of this study were three-fold: (1) to further evaluate the face validity of

DAT +/- mice as an animal model of ADHD using the 5-choice continuous performance task (5-CCPT) (Young, Light, Marston, Sharp, & Geyer, 2009). This task adds a signal detection component to the 5-choice serial reaction time task (5-CSRTT) of sustained visual attention by adding a go-no go component in which of 80% of the trial are go trials and 20% of the trials are no go trials. (2) to confirm predictive validity of DAT +/- mice as a model of ADHD by examining the effects of amphetamine in the 5-CCPT. (3) to test the hypothesis that the serotonin 1B antagonist SB224289 might ameliorate deficits in this task, and by implication might be a potential ADHD treatment.

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2.0 Methods and materials

2.1 Subjects

Male DAT +/+ mice (N = 10) and male DAT +/- mice (N = 8) were studied in the

5-CCPT. Testing occurred between 2 and 4 months of age. This DAT KO strain is the one originally developed by Ichiro Sora (Sora et al., 1998). A colony of these mice was established at the University of Toledo in 2016. Mice for these experiments were produced by crossing female DAT +/+ and male DAT +/- mice to avoid potential effects of aberrant maternal care by DAT +/- dams (Spielewoy et al., 2000). The mice were housed in a standard light cycle 12/12 hrs (lights on at 7am), standard temperature (20-22

°C), and standard humidity (40-60%). At weaning mice tissue samples for genotyping were taken by ear punches, and ear tags attached for identification. Mice were genotyped by PCR for DAT genotypes as described in previous publications (Perona et al., 2008).

All mice were socially housed, 3-4 per cage, with food and water available ad libitum prior to training. Dietary restriction was initiated two days before the first training session and body weight maintained was maintained at above 90% of their original weight.

Regular Food pellets (Teklad rodent diet 2916) moistened with strawberry flavored milk shake (Ensure®) were given to reduce neophobia, as the strawberry flavored milk shake was used as the reinforcer in operant training and testing. All experiments were conducted in accordance with all applicable guidelines for animal research, including those of the Association for Assessment and Accreditation of Laboratory Animal Care and the National Institutes of Health (NIH, USA) under protocols approved by the

University of Toledo Institutional Animal Care and Use Committee.

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2.2 Apparatus

All experiments were performed in 9-hole operant chambers (5 open holes; 19 x 24 x

22 cm; Harvard apparatus) as shown in Figure 1, controlled by Packwin software installed on a laptop computer. Each chamber consisted of an array of five circular nose- poke holes 1.3 cm diameter x 1 cm depth arranged horizontally on a curved wall 2.5 cm above the grid floor opposite a liquid reinforcer delivery magazine (four alternate holes were closed to make 5 equally-spaced holes available). The curved wall made each hole equidistant from the magazine on the opposite wall. A house-light (24V, 0.1W) was located a above the magazine. Each chamber was in a sound and light attenuating box to minimize any distraction that might affect task performance. Mice were trained to respond with a nose-poke to a lighted LED recessed into the holes. Responses were detected by photocell detector mounted vertically located 0.3 cm from the opening of the hole. Liquid reinforcement was strawberry flavor milkshake (Ensure®, 50 µl). Magazine entries were also monitored using a photocell detector.

2.3 Training protocol: 5-Choice Continuous Performance Test (5-CCPT)

Based on previous a version of the 5-CCPT (Young et al., 2009), a modified version of the 5-CCPT was developed that allowed much faster acquisition of the task, within 10 days. This modified task used long overnight daily training sessions beginning at 6 PM and ending at 6 AM every day, targeting the part of day when mice are most active. As mice reached criterion for each training stage, they immediately progressed to the next stage of training, completing as many stages as possible during each session. In the next session they would start with whichever stage they had not completed on the 239

previous day. In Stage 1 mice began with training to associate the magazine with liquid food delivery. Two drops of strawberry milk shake were dispensed in the magazine to start the session. Leaving the magazine triggered a new trial and two more milk shake drops were delivered. Each mouse needed to finish twenty trials to move to next stage.

All mice completed this stage in less than 20 min. Stage 2 of training required a single nose poke to receive a reinforcer. In this stage, one of the five holes was illuminated. If the mouse poked the illuminated hole a liquid reinforcer was delivered. Once the mouse retrieved the reinforcer the next trial was initiated. One hundred completed trials were required to progress to Stage 3. All mice successfully finished this stage in the first session. The position of the illuminated hole was pseudo-randomized to avoid the development of any position preference. At the end of Stage 2 the mice learned the association between poking the illuminated hole and reinforcer delivery. Stage 3 invoked an intertrial interval (ITI; 4 s) between retrieving the liquid reward and the initiation of the next trial. If the mouse responded prematurely, before the ITI had elapsed, the ITI would reset. At the end of the ITI the cue light in one of holes would be lit (on a pseudo- random schedule) and remain lit for a 10 s stimulus duration (SD), followed by a 2 s limited hold (LH). If the mouse responded during the SD or LH to the correct hole

(illuminated) a reinforcer was delivered. If the mouse responded in one of the four unilluminated holes, an incorrect choice was registered. If the mouse failed to respond to any of the holes during the allotted time (SD + LH) an omission was registered. Thus, there were 3 types of errors: premature responses, incorrect responses, and omissions. If the reinforcer was not delivered, a punished timeout was initiated for 5 s during which the house light was turned on. After the 5 s timeout a new trial could be initiated by entering 240

the food magazine. Starting from Stage 3, mice had to make thirty total correct choices

(ignoring errors) and to meet an 80% accuracy in 50 trials before progressing to the next stage. In Stages 4-6 the SD duration was lowered to 5, 3, and then 2 s. The criteria for completion of each stage was completion of 50 trials with 30 correct choices and less than 8 incorrect choices. After meeting the criteria at a SD of 2 s, a go/no go component was introduced in Stage 7. The initial ratio of no go to go trials was 3/2. Go trials were the same as in the previous sessions, with a SD of 2 s. In the no go trials, all holes were lit and the animal had to refrain from responding to receive the reinforcer. Errors on the go trials were thus classified as incorrect responses, premature responses and omissions, while on the no go trials, errors were classified as premature responses and false alarms

(responding when a response should be withheld). The ratio of no-go trials to go trials was gradually reduced from 3/5 to 1/5 when the false alarm ratio was less than 0.25.

After completion of this training regimen, performance in the 5-CCPT was maintained in daily test sessions of a shorter 30 min duration where the SD was 2 s, the ITI was fixed to

4 s, the no go to go trial ratio was 1/5, and the number of trials performed was unlimited within the duration of 30 minutes. Challenge sessions, described below, were started after a stable baseline was established for the 30-minute test session. Baseline sessions were given between challenge sessions to maintain performance.

2.4 5-CCPT Challenge sessions

In order to challenge sustained visual attention and motor/action impulsivity after baseline behavior was established in the 5-CCPT, mice were subjected to a challenge protocol consisting of eight different trial types. In the challenge sessions there were 241

counterbalanced trials in which the ITI and SD were varied: ITI - 4 s and 8 s; SD 0.5, 1, and 2 s. The total number of trials in each challenge session were 200, and the order of presentation was pseudo-randomized. Pharmacological treatments were given before challenge sessions once per week, with daily 30-min baseline 5-CCPT sessions conducted between each treatment.

2.5 Drug Treatments

Treatments were administered during the challenge sessions in a counterbalanced manner. As mentioned before, the mice were tested on challenge sessions once weekly.

Drugs were administered 5 minutes prior to testing. D-Amphetamine (AMPH; Sigma, St.

Louis, Mo) was dissolved in sterile saline (0.9% w/v NaCl) in doses of 0.3, 0.66, and 1.5 mg/kg, IP in a volume of 10 ml/kg). Mice were tested at weekly intervals, with baseline sessions conducted on a daily basis. After completion of these amphetamine challenge sessions, mice were not tested for 3 days during which they had free food and water.

After this time mice were retrained for 7 days to reestablish baseline performance on the

30 min 5-CCPT procedure prior to testing with the 5-HT1B antagonist SB 224289 (1'- methyl-5-[[2'-methyl-4'-(5-methyl-1,2, 4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6,7- tetrahydro-spiro[furo[2,3-f]indole-3,4'-piperidine]; Santa Cruz Biotech). SB 224289 was dissolved in 10% β hydroxypropyl cyclodextrin in distilled water, and tested at doses of

0, 10, and 20 mg/kg IP in a volume of 10 mL/kg administered 5 min before testing (based on a previous study in DAT -/- mice (Hall, Sora, et al., 2014)). Challenge tests were again given once per week with baseline testing in-between challenge tests.

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2.6 Statistics and data analysis

The data were analyzed using Graphpad Prism 8.0 software. Multifactorial repeated- measures analysis of variance (ANOVA) was used to analyze behavioral performance in terms of variables representing different aspects of performance: discriminative response accuracy (correct choices/(correct choices +incorrect choices) x 100), correct % ((correct choices/total trials) x 100), incorrect % ((incorrect choices/total trials) x 100), omission %

((omissions/total trials) x 100), premature error % ((premature errors/total trials) x 100), incorrect reaction time (defined as average latency to nose poke in the incorrect hole after stimulus presentation), reward latency (time taken to enter the food magazine after a correct response), number of false alarm rate (number of inappropriate responses during no-go trial/total no-go trials). Omission errors occur due to attentional failures, which also contribute to incorrect responses. Premature errors result from failure of response inhibition (impulse control). Two additional measures were calculated. The sensitivity index (SI; see equation 1), which reflects the ability to distinguish between go and no-go trials using correct responses on go trials and correct withholding of responses on no-go trials. The response index (RI; see equation 2), which reflects the tendency to respond to a stimulus, as opposed to not responding.

푝(퐻푖푡)−푝(퐹퐴) Equation 1. 푆퐼 = (Young et al., 2009) 2(푝(퐻퐼푡)+푝(퐹퐴))−(푝(퐻푖푡)+푝(퐹퐴))2

푝(퐻푖푡)+푝(퐹퐴)−1 Equation 2. 푅퐼 = (Young et al., 2009) 1−{푝(퐹퐴)−푝(퐻푖푡)2}

The factors in the ANOVA included the between-subjects factor of GENOTYPE

(DAT +/+ vs. DAT +/-), and the within-subjects factor of SD (0.5, 1 and 2 s), ITI (4 vs 8 s), and DOSE for AMPH or SB 224289. In the case of a significant Mauchly's Test of 243

Sphericity result, data were adjusted using a Greenhouse–Geisser correction of degrees of freedom to provide more conservative estimates of significance. Whenever applicable, significant ANOVA results were subsequently evaluated using post hoc Bonferroni means comparisons. The alpha level was set at p<0.05. Data are presented as mean ±

SEM.

3.0 Results

3.1 Amphetamine effects on behavioral performance in the 5-CCPT

Accuracy was affected by several factors including SD, as shown in Figs. 1A-C.

Accuracy was reversed at shorter SD. This was confirmed by a significant main effect of

SD in the omnibus ANOVA (F (2, 32) = 62.3, p< 0.001). Post hoc comparisons showed that SD has direct relationship with accuracy (SD 2s accuracy > SD 1s accuracy, p< 0.01;

SD 1s accuracy > SD 0.5 s, p< 0.0001) and also by ITI which was confirmed by a significant main effect of ITI (F (1, 16) = 21.4, p<0.001) and significant post hoc comparisons (p<0.001). The main effect of GENOTYPE was significant in an omnibus

ANOVA (F (1, 16) = 6.0, p<0.05), confirming that DAT+/+ mice had a higher overall accuracy than DAT +/- mice (p<0.05). Accuracy was significantly affected by amphetamine DOSE overall (F (3, 48) = 3.4, p<0.05), but post hoc comparisons showed that the 1.5 mg/kg amphetamine dose had higher accuracy than the 0.3 mg/kg amphetamine dose but not saline (p<0.05). None of the interaction terms in the ANOVA were statistically significant: ITI x GENOTYPE (F (1, 16) = 2.5, NS), SD x GENOTYPE

244

(F (2,32) = 2.2, NS), amphetamine DOSE x GENOTYPE (F (3, 48) = 1.7, NS), SD* amphetamine DOSE (F (6, 96) = 1.48, Figure 1

NS), and ITI x SD x amphetamine DOSE

(F (6, 96) = 0.6, NS).

The correct % was affected by several factors, including SD as shown in

Figs. 2 A-C. The correct % was strongly reduced at shorter SD (comparing across

Figs. A, B and C) which was confirmed by a significant main effect of SD in the omnibus ANOVA (F (2, 32) = 286.3, p<

0.0001). Post hoc comparisons confirmed these findings. The correct % at SD 2 s was greater than the correct % at SD 1 s

(p< 0.01). The correct % at SD 1 s was higher that the correct % at SD 0.5 s (p< Figure 8. the effects of amphetamine 0.0001). The correct % was also reduced (saline, 0.3, 0.66 and 1.5 mg/kg IP) on accuracy in DAT +/+ and DAT +/- mice at the longer ITI (8 s) compared to the at ITI 4 and ITI 8, and SD 2 (A), SD 1 (B), and (C) SD 0.5. shorter ITI (4 s), which was confirmed by a significant main effect of ITI in the omnibus

ANOVA (F (1, 16) = 18.8, p<0.001). The correct % was not significantly affected by

GENOTYPE, which was confirmed in the omnibus ANOVA (F (1, 16) = 0.07, NS).

Correct % was significantly affected by amphetamine DOSE (F (3, 48) = 4.25, p<0.05).

None of the interaction terms were significant. To identify the effects seen int the 245

omnibus ANOVA, separate ANOVA were performed for data for each SD. Analysis of the

SD 2 s data found a significant main effect of amphetamine DOSE (F (2.5, 74.7) = 10.5,

P<0.01). Overall comparisons for dose showed that the 1.5 mg/kg amphetamine dose was significantly increased correct % in Figure 2 comparison with saline (p<0.05). The A Correct % SD2 main factor of GENOTYPE was not 100 ✱ DAT+/+ ITI 4 90 DAT+/- ITI 4 80

% DAT+/+ ITI 8 significant (F (1, 16) = 0.32, NS). The t 70

c DAT+/- ITI 8

e r

r 60 o longer (8 s) ITI reduce correct % c 50 40 30 relative to shorter ITI (4 s) (F (1, 16) = saline .3 .66 1.5 B Amphetamine dose mg/kg

9.6, P<0.01). For the 2 s SD data none 100 Correct % SD1 90 80

of the interaction terms were %

t 70

c

e r

r 60 o significant. For the 1 s SD data, c 50 40 30 ANOVA found a significant effect of saline .3 .66 1.5 Amphetamine dose mg/kg ITI (F (1, 16) = 12.9, P<0.01), but the C 100 Correct % SD0.5 90

main effects of GENOTYPE and 80 %

70

t c

e 60 r

TREATMENT were not significant (F r

o 50 c 40 (1,16) = 0.131, NS; F (3, 48) = 1.98, 30 20 saline .3 .66 1.5 NS; respectively), nor were any of the Amphetamine dose mg/kg Figure 9.the effects of amphetamine (saline, interaction terms. For the 0.5 s SD data 0.3, 0.66 and 1.5 mg/kg IP) on accuracy in DAT +/+ and DAT +/- mice at ITI 4 and ITI ANOVA found a significant effect of 8, and SD 2 (A), SD 1 (B), and (C) SD 0.5. *p<0.05 ITI (F (1, 16) = 14.9, P<0.01), but the

246

main factors of GENOTYPE and TREATMENT were not significant (F (1, 16) = 0.06,

NS; F (3, 48) = 1.3, NS; respectively) and none of the interaction terms were significant.

The incorrect % was affected by several factors, including SD, as represented in

Figs. 3 A-C. The incorrect % increased at lower SD, which was confirmed by a

Figure 3 significant main effect of SD in the

omnibus ANOVA (F (2, 32) = 33.4,

p<0.0001). Post hoc comparisons

confirmed these findings, showing

that the incorrect % at an SD of 2 s

was much lower than the

incorrect % at the SD of 1 s

(p<0.01). The % incorrect was much

lower at an SD of 1 s than at an SD

of 0.5 s (p< 0.0001). The incorrect %

was also increased overall at the

longer ITI. This was confirmed by a

significant main effect of ITI in the

omnibus ANOVA (F (1, 16) = 22.5,

p<0.001). DAT +/- mice had a

higher incorrect % than DAT +/+ Figure 10. the effects of amphetamine (saline, 0.3, 0.66 and 1.5 mg/kg IP) on incorrect % in DAT mice, although this effect was +/+ and DAT +/- mice at ITI 4 and ITI 8, and SD 2 (A), SD 1 (B), and (C) SD 0.5. substantially impacted by the other factors. Confirming this overall picture, in the omnibus ANOVA there was a significant 247

main effect of GENOTYPE (F (1, 16) = 4.8, p<0.05). Incorrect % was not significantly affected by amphetamine DOSE overall (F (3, 48) = 2.0, NS), and no other interactions were statistically significant.

It is interesting to speculate about the nature of these increases in incorrect responses. The increases in incorrect responses could simply be a failure to notice the cue position after its Figure 4 appearance, in A DAT+/+ DAT+/- B

3.4 3.4 which case the 3.2 saline 3.2 3.0 0.3 mg/kg 3.0 latency of 0.66 mg/kg 2.8 2.8 1.5 mg/kg incorrect 2.6 2.6 2.4 2.4

responses 2.2 2.2 )

) 2.0 2.0

s

s

(

(

y

y 1.8 1.8 c

would be likely c

n

n e

e 1.6 1.6

t

t

a

a L to be higher L 1.4 1.4 1.2 1.2 than the SD 1.0 1.0 0.8 0.8 duration. 0.6 0.6 0.4 0.4 However, 0.2 0.2 0.0 0.0 another Amphetamine dose mg/kg Amphetamine dose mg/kg possibility is Figure 11. incorrect latency distribution difference between A) DAT+/+ and B) DAT+/- male mice and the effect of amphetamine. that there could be an increased number of incorrect responses at very short latencies, which basically reflect premature responses occurring just after the stimulus onset, but before there is careful observation of the stimulus, so that these responses are really more reflective of impulsive responses. There were no overall differences in incorrect choice latencies in an 248

omnibus ANOVA associated with the main effects of GENOTYPE (1, 11) = 1.0, NS), SD

(2, 22) = 3.2, NS), or amphetamine DOSE (3, 33) = 1.9, NS), nor were there any significant interactions: DOSE x GENOTYPE (F (3, 33) = 1.4, NS), ITI x GENOTYPE

(F (1, 11) = 1.2, NS), or SD x GENOTYPE (F (2, 22) = 1.1, NS). Nonetheless, examination of the distribution of the latencies was revealing. As can be seen in Fig. 4A compared to Fig. 4B, the DAT +/- mice had a cluster of very short latency incorrect responses (<0.5 s), that likely Figure 5 reflect premature, impulsive A Premature % ✱✱ 30 choices, occurring prior to ✱✱ DAT+/+ITI 4

% DAT+/- ITI 4

20

e r

sufficient time for full u

t DAT+/+ ITI 8

a m

e DAT+/- ITI 8 r 10 consideration of the appropriate p

0 response to stimuli that is saline 0.3 0.66 1.5 Amphetamine dose mg/kg necessary for accurate B responding (for discussion of the perseveration % 50 DAT+/+

relationship of response latency 40 n

o DAT+/-

i t

a 30

r

e v

to impulsive choices see e

s 20

e

r P Fitzpatrick et al. (2017)). 10 0 saline 0.3 0.66 1.5 Consistent with this analysis of Amphetamine dose mg/kg incorrect response latencies is Figure 12. the effects of amphetamine (saline, 0.3, 0.66 and 1.5 mg/kg IP) on % premature responses the analysis of the premature A) and % perseveration (B) in DAT +/+ and DAT +/- mice at ITI 4 and ITI 8. **p<0.01 responses.

Premature errors (expressed as the percentage of premature responses; premature

%) were significantly increased at the longer ITI, consistent with previous observations in 249

the 5-CSRTT (Dalley et al., 2007; Dalley, Mar, Economidou, & Robbins, 2008; Huang et al., 2017). This was confirmed by a significant effect of ITI in the ANOVA (Fig. 5A; F

(1, 14) = 69.2; p<0.0001). There was a significant post hoc difference in premature errors between DAT +/+ and DAT +/- mice at the long ITI (p<0.01). Premature responses were Figure 6 greater in DAT +/- mice than in

DAT +/+ mice overall as shown by A 80 Omission % SD2 DAT+/+ ITI 4 ✱✱✱✱ a main effect of GENOTYPE (F (1, 60

% DAT+/- ITI 4

n

o i

s 40 DAT+/+ ITI 8

s i

14) = 5.5; p<0.05). Amphetamine m O 20 DAT+/- ITI 8

DOSE reduced premature responses 0 saline .3 .66 1.5 overall in the ANOVA (F (1, 15) = Amphetamine dose mg/kg

3.3, p<0.05), but these differences B 80 Omission % SD1 were primarily observed in DAT +/-

60

%

n

o i

mice at the long ITI (Fig. 5A). s 40

s

i

m O These mice had the most premature 20 0 responses, and post hoc saline .3 .66 1.5 Amphetamine dose mg/kg C comparisons showed that 1.5 mg/kg 80 Omission % SD.5

amphetamine reduced premature 60

%

n

o i

s 40 s

response compared to saline i m

O 20 treatment in DAT +/- mice at the 0 saline .3 .66 1.5 long ITI (p<0.001). This overall Amphetamine dose mg/kg pattern of effects was also Figure 13. the effects of amphetamine (saline, .3,0.66, and 1.5 mg/kg IP) on omission% in confirmed by significant DOSE x DAT+/+ and DAT+/- male mice at ITI4 s and ITI 8 s and SD 2 s(A), SD 1 s(B), and SD 0.5 s ITI (F (3, 42) = 3.5; p<0.05), and (C). ****p<0.0001 250

ITI x GENOTYPE (F (1, 15) = 5.7, p<0.05) interactions, but the interactions of DOSE x

GENOTYPE (F (3, 45) = 1.1, NS), and DOSE x ITI x GENOTYPE were not significant

(F (3, 45) = 2.1, NS) .

The effect of amphetamine dose on perseverative responses (perseveration %) are presented in Figure 5B. Although there was a trend for more perseverative errors in DAT

+/- mice, there were not significant effects of GENOTYPE (F (1, 15) = 4.5, NS), amphetamine DOSE (F (2.4, 37.3) = 0.8, NS), or GENOTYPE x amphetamine DOSE (F

(3, 45) = 1.4, NS) in the ANOVA.

The effects of DAT Figure 7 GENOTYPE and amphetamine DOSE A Senstivity index

0.8

on omissions, expressed as ✱ DAT+/+ITI 4 x

e 0.6

d DAT+/- ITI 4

n

i

y t omission %, are represented in Figs. i 0.4

v DAT+/+ ITI 8

i

t

s n

e 0.2 DAT+/- ITI 8 3A-C. Omissions were increased at S 0.0 saline 0.3 .66 1.5 shorter SD, which was confirmed by a Amphetamine dose mg/kg significant main effect of SD in the B omnibus ANOVA (F (2, 32) = 229.6, Response index

0.4 x

p<0.0001). Overall comparisons e 0.2

d

n

i

e

s 0.0

n o

confirmed an increase in omissions at p s

e -0.2 r lower SD values (SD 0.5 s omission % -0.4 saline .3 .66 1.5 Amphetamine dose mg/kg > SD 1 s omission %, p<0.01; SD 1 s Figure 7. The effects of amphetamine (saline, omission % > SD 0.5 s omission %, 0.3, 0.66 and 1.5 mg/kg IP) on the sensitivity index A) and the response index (B) in DAT +/+ p<0.0001). Omissions were also and DAT +/- mice at ITI 4 and ITI 8. *p<0.05 affected by ITI, which was confirmed by a significant main effect of ITI (F (1, 16) = 10.0, 251

p<0.01), although there was a much smaller effect of ITI on omissions than SD on omissions. There was no overall effect of GENOTYPE on omissions (F (1, 16) = 0.4,

NS), but amphetamine did reduce omissions under some conditions, primarily at SD 2, which was shown by a significant main effect of amphetamine DOSE (F (3, 48) = 3.1, p<0.05). No other interactions were statistically significant in the ANOVA. The effect of amphetamine was confirmed by post hoc Figure 8 A comparisons showing that in the SD 2 s, there Accuracy SD 2 100 DAT+/+ ITI 4

90 % was overall decrease in omission % by 1.5

y 80 DAT+/- ITI 4

c

a

r u

c 70 DAT+/+ IT I8

c a mg/kg amphetamine compared to saline in all 60 DAT+/-ITI 8 50 groups (p<0.0001). Vehicle 10 20 SB224289 dose mg/kg Amphetamine DOSE increased the

B Accuracy SD 1 sensitivity index in the 5-CCPT (F (3, 48) = 100

90

%

4.5; p<0.05), but as is clear from Fig. 7A, the y 80

c

a

r u

c 70

c A increase in the sensitivity index was seen in 60

50 Vehicle 10 20 DAT +/+ mice, but not DAT +/- mice. This SB224289 dose mg/kg was confirmed by post hoc comparisons C Accuracy SD 0.5

100

(p<0.05 vs. saline) which were significant in 90

%

y 80

c

a r

DAT +/+ mice at the short ITI, but not at the u

c 70

c A 60 long ITI in DAT +/+ mice, and at neither ITI 50 Vehicle 10 20 SB224289 dose mg/kg in DAT +/- mice. No significant overall Figure 8. the effects of SB 224289 differences were found for GENOTYPE (F (vehicle, 10 and 20 mg/kg IP) on accuracy in DAT +/+ and DAT +/- mice (1, 16) = 0.0, NS). A significant main effect at ITI 4 and ITI 8, and SD 2 (A), SD 1 (B), and (C) SD 0.5. of ITI was found (F (1, 16) = 17.4, p<0.001), 252

consistent with a reduced sensitivity index at lower ITIs. None of the other interactions in the ANOVA were statistically significant.

The response index appeared to increase in response to amphetamine, but these effects were not significant (F (3, 48) = 2.7, NS). Response index was not significantly affected by any of the other factors.

There were no effects on reward latency resulting from any of the factors, including GENOTYPE and amphetamine DOSE (data not shown). This indicates that there were no differences in motivation for the food rewards resulting from these factors that might constitute a confounding influence.

3.2 Effects of SB 224289 on behavioral performance in the 5-CCPT

The overall pattern of effects in the 5-CCPT for SD, ITI, and DAT genotype were similar to those observed in the amphetamine study. In the SB 224289 data, accuracy was affected by SD in a similar manner to the previous study and was reduced at lower SD

(Fig. 8). This was confirmed by a significant main effect of SD in the omnibus ANOVA

(F (2, 32) = 45.8, p<0.001). Post hoc comparisons showed that accuracy differed between

SD conditions: Overall comparisons of SD data showed that SD 2 s accuracy > SD 1 s accuracy, p< 0.01; SD 1 s accuracy > SD 0.5 s accuracy, p<0.0001. Again, ITI had a much smaller, but significant overall effect on accuracy (F (1,16) = 5.1, p<0.05).

GENOTYPE did not significantly affect accuracy overall (F (1, 16) = 0.6, NS), or did SB

224289 DOSE (F (3, 48) = 0.4, NS), nor were any of the interactions in the ANOVA significant.

253

Figure 9 Correct % was reduced at lower SD,

A as was observed previously in the 100 Correct % SD 2 DAT+/+ ITI4

80 amphetamine study (Fig. 9). This was

%

t 60 DAT+/-ITI 4

c

e r r 40 o DAT+/+ ITI8

C confirmed by a significant main effect of SD 20 DAT+/- ITI 8 0 Vehicle 10 20 in the omnibus ANOVA (F (2, 32) = 213.9, SB224289 dose mg/kg p<0.0001). Post hoc comparisons between B Correct % SD 1 100 the SD conditions showed that Correct % ✱

80

%

t 60 differed with SD: SD 2 s Correct % > SD 1 s

c

e r

r 40 o C Correct %, p<0.001; SD 1 s Correct % > SD 20

0 Vehicle 10 20 0.5 s Correct %, p<0.0001). In this study the SB224289 dose mg/kg C effect of ITI was not significant (F (1, 16) = 100 Correct % SD 0.5

80 2.2, NS). GENOTYPE did not significantly

%

t 60

c

e r r 40

o affect Correct % (F (1, 16) = 1.9, NS), but C 20

0 there was a significant overall effect of SB Vehicle 10 20 SB224289 dose mg/kg 224289 Dose on Correct % (F (2, 32) = 6.6, Figure 9. the effects of SB 224289 (vehicle, 10 and 20 mg/kg IP) on correct % in DAT p<0.05). As can be seen in Fig. 9, there were +/+ and DAT +/- mice at ITI 4 and ITI 8, and SD 2 (A), SD 1 (B), and (C) SD 0.5. trends for reductions in Correct % which were strongest at the lower ITI and lower SD conditions. None of the interaction terms in the ANOVA were significant.

To further analyze these effects on Correct %, individual ANOVA were performed on the data from each SD. No differences were found for SD 0.5 or SD 2, but at SD 1 SB 224289 significantly decreased correct responses (F (2, 30) = 4.7, p<0.05),

254

which was significant at the 20 mg/kg dose (p<0.05) in DAT +/+ mice, but not DAT +/- mice.

As in the previous experiment, Figure 10 incorrect % increased with increasing A

20 SD values (Fig. 10), which was Incorrect% SD2 DAT+/+ ITI 4 DAT+/-ITI 4

15 %

DAT+/+ ITI 8

t

c e

confirmed by a significant main effect r 10 DAT+/-ITI 8

r

o

c

n i of SD in the omnibus ANOVA (F (2, 5

0 Vehicle 10 20 32) = 41.2, p<0.0001). Comparisons of SB224289 dose mg/kg the incorrect % between SD conditions was consistent with these overall B

20 findings, showing: SD 0.5 s Incorrect SD1

15

%

t

incorrect % > SD 1 s incorrect %, c e

r 10

r

o

c n p>0.05; SD 1 s incorrect % > SD 2 s i 5

0 incorrect %, p< 0.0001. ITI did not Vehicle 10 20 SB224289 dose mg/kg significantly affect incorrect Y (F (1, C

20 16) = 0.4, p>0.05), nor were there Incorrect SD0.5

15

%

t

c e

significant main effects of r 10

r

o

c

n i GENOTYPE (F (1, 16) = 2.0, NS) or 5

0 Vehicle 10 20 SB 224289 DOSE (F (3, 48) = 2.4, SB224289 dose mg/kg

NS), and none of the interactions were Figure 10. the effects of SB 224289 (vehicle, 10 and 20 mg/kg IP) on incorrect % in DAT significant either. +/+ and DAT +/- mice at ITI 4 and ITI 8, and SD 2 (A), SD 1 (B), and (C) SD 0.5. Premature responses were again greater and the longer ITI and DAT +/- mice had more premature responses, e.g. 255

higher premature %, than DAT +/+ mice (Fig. 11B). SB224289 significantly reduced premature responses as shown by a significant effect of SB 224289 DOSE; (F (2, 30) =

6.7, p<0.005). Post hoc comparisons demonstrated that premature responses were significantly reduced by SB 224289 in DAT +/- mice at the 10 and 20 mg/kg doses

(p<0.05 and p<0.001, respectively), but in DAT +/+ mice, which had lower levels of premature responses to start with. Figure 11 Effects of DAT genotype and

A SB224289 treatment on omissions, expressed Sensitivity index 0.6

DAT +/+ ITI 4

x e

as omission %, are represented in Fig. 12. As d 0.4

n i

DAT +/- ITI 4

y

t

i

v

i t

s DAT+/+ ITI 8

0.2 n

before, omissions increased dramatically as e S

DAT +/- ITI 8 0.0 the SD increased, which was confirmed by a Vehicle 10 20 SB224289 dose mg/kg significant main effect of SD in the omnibus B ✱✱ ✱ 25 ANOVA (F (2, 32) = 200.2, p<0.0001).

20

%

e

r 15

u t Comparisons of the omissions at the different a

m 10

e

r P 5 SD values clearly showed this pattern: SD 0.5 0 Vehicle 10 20 s omission % > SD 1 s omission %, p<0.01; SB224289 dose mg/kg C response index

0.4

SD 1 s omission % > SD 2 s omission %, p< x

e 0.2

d

n

i

e

s 0.0

0.0001. ITI did not significantly affect n

o

p s

e -0.2 omission % (F (1, 16) = 1.3, NS), nor did R -0.4 Vehicle 10 20 GENOTYPE (F (1, 16) = 2.5, NS). However, SB224289 dose mg/kg

SB 224289 did increase omission %, although Figure 11. The effects of SB 224289 (vehicle, 10 and 20 mg/kg IP) sensitivity this was affected somewhat by genotype. index (A), premature responses (B) and response index (C) in DAT +/+ and DAT Overall, omission % was increased by SB +/- mice at ITI 4 and ITI 8 256

224289, shown by a significant effect of Figure 12

SB 224289 DOSE (F (3, 48) = 7.8, 80 Omission SD2 DAT+/+ ITI4

p<0.01), but no other interactions were 60 %

DAT+/- ITI 4

n o

i 40

s s i DAT+/+ ITI8 statistically significant in the omnibus m O 20

DAT+/- ITI 8 ANOVA. Nonetheless, it is clear that 0 Vehicle 10 20 SB224289 dose mg/kg under several conditions, the

80 omission % was increased by SB Omission SD1

224289 in DAT +/+ mice, but not in 60

%

n o

i 40

s

s i

DAT +/- mice m O 20

The sensitivity index (Fig. 11A) and 0 Vehicle 10 20 response index (Fig. 11C) were not SB224289 dose mg/kg significantly affected by any factor, 80 Omission SD0.5

including SB224289 DOSE. 60

%

n o

i 40

s

s i

There were no effects on reward m O 20 latency resulting from any of the factors, 0 Vehicle 10 20 including genotype and amphetamine SB224289 dose mg/kg Figure 12. the effects of SB 224289 dose (data not shown). This indicates (vehicle, 10 and 20 mg/kg IP) on omission % in DAT +/+ and DAT +/- mice at ITI 4 that there were no differences in and ITI 8, and SD 2 (A), SD 1 (B), and (C) SD 0.5. motivation for the food rewards resulting from these factors that might constitute a confounding factor.

257

4.0 Discussion

The present study examined attention and impulsivity in DAT +/- mice using the

5-CCPT. Evidence of impulsivity was found in DAT +/- mice, in terms of increased premature responding, providing evidence for face validity of DAT +/- mice as an animal model of ADHD. Measures of attention were not affected in the current study. In further validation of the model it will be important to examine these mice in other circumstances, including addressing distractibility, which was not addressed here. Additionally, amphetamine ameliorated motor impulsivity providing evidence for predictive validity of the model. Interestingly, amphetamine also increased the sensitivity index in DAT +/+ mice, consistent with some previous observations (MacQueen, Minassian, Henry, et al.,

2018; MacQueen, Minassian, Kenton, et al., 2018), but not in DAT +/- mice, which may suggest the importance of DAT in the mechanism through which amphetamine enhances vigilance in this task. Importantly, this study also showed that the serotonin 1B antagonists SB224289 also reduced motor impulsivity, in terms of premature responses, perhaps indicating that this mechanism may be useful in the treatment of impulsive conditions, including those associated with ADHD.

4.1 DAT +/- mice as a model of ADHD

This study was partly initiated to determine if DAT +/- might show symptoms of

ADHD, either impulsivity or attentional deficits, that might be used to better model

ADHD-like deficits. The DAT -/- model has been shown to have both face and predictive validity, but the extremity of the DAT -/- manipulation does not accord with differences in DAT function in humans, and in preliminary experiences we found that DAT -/- mice 258

were not even able to learn the most basic aspects of these task, failing to progress past the first training phase. Nonetheless, DAT -/- mice do show many ADHD-like deficits, including locomotor hyperactivity (Fox et al., 2013; Giros et al., 1996; Ralph et al., 2001;

Sora et al., 2001; Sora et al., 1998), deficits in prepulse inhibition of acoustic startle (PPI)

(Arime et al., 2012; Ralph et al., 2001; Wong et al., 2012; Wong et al., 2015; Yamashita et al., 2006; Yamashita et al., 2013), impairments in the cliff avoidance reaction (CAR)

(Yamashita et al., 2013), and general learning impairments (Gainetdinov et al., 1999; Li et al., 2010; Morice et al., 2007; Wong et al., 2012; Wong et al., 2015) that are likely to result as secondary consequences of other ADHD-like deficits in DAT -/- mice. In the majority of those studies DAT +/- mice were not examined, and in the few cases where they were examined, they did not show deficits.

In addition to a certain degree of superficial face validity, many of the deficits observed in DAT -/- mice can also be reversed by drugs that are effective ADHD treatments, providing evidence for predictive validity for the model. ADHD treatments, including amphetamine and methylphenidate, have been shown to reverse hyperactivity

(Gainetdinov et al., 1999), PPI deficits (Yamashita et al., 2006), and CAR deficits

(Yamashita et al., 2013) in DAT -/- mice. Additionally, consistent with the high rate of smoking in ADHD patients (van Amsterdam et al., 2018), nicotine also reduces hyperactivity and PPI deficits (Uchiumi et al., 2013) and improves learning deficits

(Weiss, Nosten-Bertrand, et al., 2007; Weiss, Tzavara, et al., 2007) in DAT -/- mice.

Another effective treatment for ADHD is atomoxetine, a selective NET inhibitor. This drug has not been assessed in DAT -/- mice, but another NET inhibitor, nisoxetine, reverse PPI and CAR deficits in DAT -/- mice (Yamashita et al., 2006; Yamashita et al., 259

2013). Indeed, direct administration of nisoxetine into the prefrontal cortex also reverses

PPI deficits in DAT -/- mice (Arime et al., 2012). Indeed, alternations in prefrontal cortex morphology and function, as well as frontostriatal function, has been repeatedly found in

DAT -/- mice (Berlanga et al., 2011; Costa et al., 2006; Cyr et al., 2003; Cyr et al., 2005;

Kasahara et al., 2015; Shen et al., 2004; Xu et al., 2009; Zhang et al., 2010). This overall picture is not only consistent with the idea that frontostriatal dysfunctions underlie

ADHD, but more specifically that noradrenergic dysfunctions associated with frontostriatal impairments underlie the disorder (Biederman & Spencer, 1999), and perhaps the actions of many effective ADHD treatments.

The DAT -/- model has not been assessed with approaches that more specifically address aspects of higher cognition, including attention, decision making and executive function. This is likely one of the instances in which unpublished findings are quite telling – in preliminary studies for the experiments presented DAT -/- mice were found to be incapable of learning even the most basic training criteria for the 5-CCPT (Saber and

Hall, unpublished findings). This may be consistent with the rare incidence of complete

DAT deletions in humans as well as the much more severe phenotypes associated with that condition (Hansen et al., 2014). This raises a fundamental problem in modeling complex polygenic disorders such as ADHD (Demontis et al., 2019; Faraone & Larsson,

2019) with single gene mutations in mice: individual mutations that are similar to what are observed in human patients may not be sufficient on their own to produce the phenotypes observed in the disease. It is possible that polymorphisms in many genes may ultimately contribute to alterations in DAT function in individuals with ADHD, so that partially reduced DAT function may still be sufficient to induce ADHD-like phenotypes, 260

although perhaps of a lesser magnitude than that observed in DAT -/- mice. This becomes an empirical question.

The alterations in dopamine function resulting from reduced DAT function in

DAT +/- are less severe than those resulting from complete elimination of DAT in DAT -

/- mice. Tissue dopamine content is reduced, but not as severely as in DAT -/- mice; D1 and D2 dopaminergic receptor expression is reduced, but not as much as in DAT -/- mice; and extracellular striatal dopamine levels are only about two times higher in DAT +/- mice, as compared to the ten times higher levels observed in DAT -/- mice (Giros et al.,

1996; Jones et al., 1998; Shen et al., 2004). Differences in locomotor activity were not observed in DAT +/- mice in initial studies (Giros et al., 1996; Sora et al., 1998), although it was a common observation that these levels were slightly, but not significantly, increases. Several subsequent studies did confirm that locomotor activity was slightly elevated in in DAT +/- mice (Hall, Itokawa, et al., 2014; Mereu et al., 2017), especially after repeated testing (Spielewoy et al., 2000), but the magnitude of this difference was much smaller than observed in DAT -/- mice. However, several other deficits found in DAT -/- mice are not observed in DAT +/- mice, including impaired PPI

(Mereu et al., 2017; Ralph et al., 2001), and impairments in the marble burying task (Fox et al., 2013).

Because of the extremity of the DAT -/- model, and perhaps the inability to examine certain aspects of ADH in this model, efforts have been made to explore potential ADHD-like deficits in DAT +/- mice. Impairments in novel object recognition

(Franca et al., 2016; Mereu et al., 2017), in the reversal stage of an attentional set-shifting task (Cybulska-Klosowicz, Dabrowska, et al., 2017; Cybulska-Klosowicz, Laczkowska, 261

et al., 2017), and in measures of attention and impulsivity in the 5-Choice serial reaction time task (5-CSRTT) (Mereu et al., 2017) have been observed in DAT +/- mice. In the 5-

CSRTT task DAT +/- mice were impulsive and showed some evidence of impaired attention. Amphetamine treatment normalized DAT +/- performance (Mereu et al., 2017), but it was not clear from the way that the drug was administered in that test whether the effects really resulted from amphetamine treatment or were rather the result of continued testing of the subjects. The results presented in the current study found some evidence of attentional deficits in DAT +/- mice, but most of these differences were not statistically significant. By contrast, there was substantial evidence for impulsive deficits in this task in DAT +/- mice, which included increased numbers of premature responses.

Additionally, more detailed analysis of the time latency distribution of incorrect choices showed that het DAT +/- mice has a cluster of incorrect choice latency responses between

0-0.5 s. This time window has been suggested to be insufficient to perceive the cue, process the information the input and execute a motor response in this task (Fitzpatrick et al., 2017). Indeed, it is highly possible that time perception impairments may underlie aspects of ADHD (Ptacek et al., 2019), contributing to many types of cognitive deficits in this disorder including inattention due to cognitive overload, impulsivity and poor decision making. A temporal perception impairment endophenotype associated with executive dysfunction has been observed for ADHD (Hove, Gravel, Spencer, & Valera,

2017; Hwang, Gau, Hsu, & Wu, 2010), and ADHD symptom severity can be assessed based on time processing and perceptual impairments (Pretus et al., 2018; Ptacek et al.,

2019). It is believed that time perception is regulated by dopamine systems, and several studies of animals with dopamine signaling dysfunctions might show these types of 262

deficits, primarily resulting from action impulsivity deficits (Adriani et al., 2009; De

Corte, Wagner, Matell, & Narayanan, 2019; Milienne-Petiot, Geyer, Arnt, & Young,

2017; Yamashita et al., 2013).

4.2 Predictive Validity of the DAT +/- model

The DAT -/- has been shown to have predictive validity based upon the reversal of deficits observed in that genotype. However, DAT +/- show minimal or no deficits in those circumstances. It thus becomes important to know whether deficits in DAT +/- mice can also be reversed by effective ADHD treatments. The present study found evidence of impulsive deficits in the 5-CCPT. Importantly, amphetamine normalized motor impulsivity deficits (premature responses) in the 5-CCPT. Psychostimulants, including methylphenidate and amphetamine, are the most widely used ADHD treatments and have been shown to treat many ADHD symptoms, including attention deficits and hyperactivity. In addition, studies have shown that psychostimulant drugs improve time processing and perception deficits in patients with ADHD (Fostick, 2017;

Pollak, Shomaly, Weiss, Rizzo, & Gross-Tsur, 2010; Rubia, Halari, Christakou, &

Taylor, 2009), which might help contribute to amelioration of the sorts of impulsive deficits identified in the 5-CCPT observed here in DAT +/- mice.

The conditions tested here did not identify impairments in attentional function in

DAT +/- mice, although several trends in that direction were observed. There may be several reasons for that. Among those reasons are that this study may have been slightly underpowered for resolving those effects. It may also be that the attentional impairments may be better resolved by the inclusion of a distracting stimulus, or changes in some of 263

the other parameters that affected attentional measures. The present study also examined adult mice. It is highly possible that the attentional deficits may be more pronounced in adolescent DAT +/- mice, than were observed for the adult DAT +/- mice studied here.

Finally, this study only examined male mice. In studies of other ADHD-like phenotypes substantial sex differences have been observed (Saber and Hall, unpublished findings), so it is possible that females may actually show greater attentional deficits than males, as we have seen for some other phenotypes.

4.3 SB 224289 as a potential therapeutic for ADHD

Given the argument for predictive validity of the DAT +/- model (albeit based on limited observations at this point), it should be presumed that this model could be used to evaluate novel non-stimulant approaches to the treatment of ADHD. Hall, Sora, et al.

(2014) found initial evidence that the serotonin 1B antagonist SB 224289 reduces locomotor activity in DAT -/- mice. DAT +/- mice were not assessed in that study, and, in any case, only show minor increases in locomotor activity (Hall, Itokawa, et al., 2014).

The present study found evidence of increased impulsivity in DAT +/- mice in the 5-

CCPT, and demonstrated that amphetamine would reverse these deficits. Consistent with the idea the antagonism of 5HT1B receptors might constitute a novel treatment approach to ADHD, the 5HT1B antagonist SB 224289 was found to reverse these deficits.

The locus of action of nisoxetine underlying its ability to reduce ADHD-like deficits in DAT -/- mice was found to be the prefrontal cortex. Serotonin 1B receptors are localized on both dendrites/cell bodies and at presynaptic terminals they control release of serotonin or other neurotransmitters (Peddie et al., 2008), including in the prefrontal 264

cortex. Modulation of serotonin 1B receptors is known to modulate behavior through several mechanisms that might be relevant to the effects of SB 224289 in DAT +/- mice.

Serotonin 1B antagonism increases the release of acetylcholine in the prefrontal cortex

(Hu et al., 2007), which might have consequences on attention and cognition, and has been suggested to underlie the effects of nicotine in DAT -/-mice (Uchiumi et al., 2013).

Further supporting the potential 5HT1B receptors to influence ADHD-like phenotypes,

5HT1B KO mice show greater cognitive flexibility have been suggested to be mediated by alterations in prefrontal acetylcholine release (Wolff et al., 2003). Expression of cortical 5HT1B receptors at different times of life has been shown to differentially affect hyperlocomotion, impulsivity and aggression (Nautiyal et al., 2015). Also, SB224289 facilitates learning consolidation and reverses memory and learning impairments induced by scopolamine (Gaster et al., 1998; Selkirk et al., 1998), further strengthening this 5HT- acetylcholine connection. As further evidence of the connection of 5HT1B receptors to

ADHD, genetic polymorphisms 5HT1B receptor gene (HTR1B) are associated with

ADHD in humans (Guimaraes et al., 2009; Hawi et al., 2002; Ickowicz et al., 2007; Quist et al., 2003; van Rooij et al., 2015).

Conclusions

This study provides evidence of ADHD-like impulsive deficits in DAT +/- mice.

Moreover, these deficits are ameliorated by the effective ADHD medication amphetamine. These data therefore provide initial evidence for the face and construct validity for the DAT +/- model, which may provide an improved model for studying the role of DAT dysfunctions in ADHD, and in ADHD-like cognitive phenotypes. 265

Additionally, this study provided the first evidence in this model that 5HT1B antagonisms might have beneficial effects for ADHD-like phenotypes. The current observations are limited to impulsivity, it will be important to further explore other phenotypes in DAT +/- mice, and the effectiveness of known and potential ADHD therapies in this model.

266

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282

Chapter 7

Summary: Preclinical evaluation of a potential treatment for ADHD targeting the serotonin 1B receptor subtype

Yasir Saber

Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and

Pharmaceutical Sciences, The University of Toledo, OH, USA; Ninevah College of medicine, Ninevah university, Mosul, Iraq

283

1.0 Introduction

Attention deficit/hyperactivity disorder (ADHD) is a common neuropsychiatric disorder that affects both children and adults (Fayyad et al., 2007; Lecendreux, Konofal,

Cortese, & Faraone, 2015; Polanczyk, Willcutt, Salum, Kieling, & Rohde, 2014; Visser et al., 2014). Symptom profiles often change as children with ADHD become adults with

ADHD. Similarly, the symptom profiles of females with ADHD may differ from the typical pattern seen in males (e.g. less hyperactivity), and this has consequently led to an apparent increase in diagnosis of females with ADHD (Fairman, Peckham, & Sclar,

2017). This point is important when considering animal models of ADHD, for which it has often been assumed that there should be a male bias. In reality, it seems that the nature of this bias should not be one of differential incidence between sexes as much as differential symptom profiles between sexes. These changes in apparent ADHD incidence might also reflect actual changes in ADHD incidence, perhaps due to changing environmental factors. In any case, there is continued interest in the development of better treatments for ADHD because of large costs involved, both personal and societal

(de Zeeuw, van Beijsterveldt, Ehli, de Geus, & Boomsma, 2017; Fenesy, Teh, & Lee,

2019; Rietveld & Patel, 2019; Zendarski, Mensah, Hiscock, & Sciberras, 2019;

Zendarski, Sciberras, Mensah, & Hiscock, 2017), which would certainly include the increased prevalence of co-morbid substance use disorders (SUD) in individuals with

ADHD (Biederman, Newcorn, & Sprich, 1991; Romo et al., 2018). The increased prevalence of SUD in individuals with ADHD may either reflect common underlying causative factors or perhaps the influence of early exposure to psychostimulant drugs, which are the most commonly prescribed treatments for ADHD, even in young children 284

(Gerlach, Grunblatt, & Lange, 2013; Khan et al., 2017). Thus, an important goal of ongoing research into ADHD treatments is the identification of alternative non-stimulant approaches to the treatment of ADHD.

This raises the question of best to identify potential ADHD treatments. Chapter 2 discussed the attributes of genetic models of ADHD. While environmental models certainly exist, reflecting the influence of known environmental factors (Palladino,

McNeill, Reif, & Kittel-Schneider, 2019), the high heritability of ADHD (Faraone &

Doyle, 2001; Larsson, Chang, D'Onofrio, & Lichtenstein, 2014) has led to focus upon genetic models. However, as discussed in Chapter 1, most of these genetic models have focused on hyperactivity as the primary outcome making them models of ADHD. This ignores the symptom heterogeneity observed in the disorder, the differences in symptoms in adults with ADHD, the symptoms that are most common in females with ADHD, and also, perhaps, the most important symptoms – impulsivity, attention deficit, and poor executive function/decision making. Indeed, when discussing ADHD-associated problems with parents of children with ADHD, one of the primary problems is poor decision making, or perhaps risk assessment, that leads children with ADHD to do things that are inherently dangerous (Dekkers et al., 2018; Todokoro et al., 2018).

Thus, in assessing the validity of an animal model of ADHD, it is important that it represent all of the observed symptoms, or at least it should be understood which symptoms it does represent, and which it does not. In the Diagnostic statistical manual of mental disorders (Diagnostic and statistical manual of mental disorders : DSM-5, 2013)

ADHD is divided into sub-types based on the most common symptoms: (1) inattentive,

(2) hyperactive / impulsive, and (3) combined of inattentive and hyperactive / impulsive. 285

The problem with this categorization is that it might not well represent symptom presentation in patients, and fails to recognize the full range of symptoms, and their independent presentation. Partially in response to these problems, not just for ADHD but for all psychiatric diagnoses, the National Institute of Mental Health has proposed an alternative approach to psychiatric diagnoses, called the Research Domain Criteria initiative (RDoC; see discussion in Baroni and Castellanos (2015)). This nosology is intended to resolve several problems with previous approaches to psychiatric diagnoses.

This include the issue that some symptoms occur in multiple disorders, and also that not all symptoms occur in each individual with a disorder. This is certainly true for ADHD.

Additionally, traditional approaches do not include any biological criteria for diagnoses, or for identifying effective treatments for particular patients. One of the hopes of RDoC is to incorporate biological measures into psychiatric diagnoses. In the RDoC approach, classical categorical definitions of psychiatric disorders do not exist, but they still map onto particular symptom profiles that are well known for those classically defined disorders. Thus, ADHD maps onto several functionally defined RDoC domains that include reward-related processing, inhibition, vigilant attention, reaction time variability, timing and emotional liability. These symptom profiles generally match the primary symptoms defined in DSM 5, including hyperactivity and impulsivity, but also include a wider range of cognitive and affective changes such as impaired executive function and decision making (Pineda-Alhucema, Aristizabal, Escudero-Cabarcas, Acosta-Lopez, &

Velez, 2018).

In any case, in considering animal models of ADHD, it is essential to consider the full range of symptoms present, which are not terribly comprehensive in DSM 5. Put as 286

simply as possible this would be attentional impairment, hyperactivity, impulsivity, and deficits in executive function. One of the key issues with current treatments is that not all symptoms are equally treated, which is perhaps not surprising if the animal models that are used to identify such treatments really only address the symptom of hyperactivity, which is not present in all individuals with ADHD. The majority of children with ADHD

(69%) are prescribed medication, primarily the psychostimulant drugs methylphenidate and amphetamine (Punja et al., 2016; Storebo et al., 2015). Some non-psychostimulant medications are available for the treatment of ADHD, including atomoxetine, guanfacine and clonidine, but many patients do not respond to these treatments (Beherec et al.,

2014), and side effects are a major concern, as they are for psychostimulant treatment approaches. Added to these concerns would be the failure to effectively treat all ADHD symptoms, especially cognitive symptoms.

As suggested here, the focus on hyperactivity in animal models of ADHD

(Carvalho, Vieira Crespo, Ferreira Bastos, Knight, & Vicente, 2016; Wickens, Hyland, &

Tripp, 2011) may limit the ability of those models to identify better therapeutics, particularly for the cognitive symptoms of ADHD. One proposed ADHD model, dopamine transporter knockout (DAT KO) mice (Arime, Kubo, & Sora, 2011;

Gainetdinov et al., 1999), show a fuller range of ADHD symptoms that includes hyperactivity (Arime et al., 2011; Giros, Jaber, Jones, Wightman, & Caron, 1996; Sora et al., 1998), impaired sensorimotor gating (a preattentional process) (Arime, Kasahara,

Hall, Uhl, & Sora, 2012; Ralph, Paulus, Fumagalli, Caron, & Geyer, 2001; Wong et al.,

2012; Wong, Sze, Chang, Lee, & Zhang, 2015; Yamashita et al., 2006; Yamashita et al.,

2013), impaired executive function or impulsivity (Yamashita et al., 2013), and deficits in 287

tests of learning and cognitive performance (Del'Guidice et al., 2014; Dzirasa et al., 2009;

Gainetdinov et al., 1999; Li, Arime, Hall, Uhl, & Sora, 2010; Morice et al., 2007; Wong et al., 2012; Wong et al., 2015). Although learning impairments have been shown in DAT

KO mice, assessment of complex tasks measuring attention, impulsivity, and aspects of executive function have not been extensively studied.

The experiments presented in this thesis were intended to accomplish several purposes: 1) to extend studies of DAT KO mice as an animal model of ADHD by more broadly examining the consequences of DAT deletion, including examination of DAT +/- mice and female mice; 2) to examine DAT KO mice in models of higher cognitive function that might better represent the sort of attentional and impulsive deficits observed in people with ADHD; and 3) to use the model to examine 5HT1B antagonism as a potential therapeutic approach to the treatment of ADHD.

2.0 The DAT KO model of ADHD: Isolation rearing

One of the problems with the DAT KO model is that the symptoms are observed after complete deletion of the DAT gene, but few symptoms are observed in DAT +/- mice that might better represent the range of variation in DAT function seen in humans.

Additionally, sex has not been considered as a factor in most studies of DAT KO mice.

Many of the studies reported in this thesis addressed these questions.

In terms of developing a better DAT KO model based on DAT +/- mice, the first experiments (Chapter 3) attempted to potentiate the small effects observed under some circumstances in DAT +/- mice (Hall, Itokawa, et al., 2014) by adding an additional environmental factor known to produce ADHD-like deficits, isolation rearing (Hall et al., 288

1998; Wilkinson et al., 1994). This series of studies examined the interaction between isolation rearing, social isolation after weaning, and partial elimination of the DAT gene in DAT +/- mice. However, the combination of isolation rearing and partial DAT deletion in DAT +/- mice did not increase the severity of the deficits observed in the presence of either factor alone. Indeed, in some behavioral tests the two factors appeared to counteract each other. There is no ready explanation for these results based on the present data. However, one possibility might be that DAT +/- are less sensitive to the effects of social isolation, perhaps because it alters social play, the critical experiential mediator of the effects of isolation rearing ((Einon, Morgan, & Kibbler, 1978); see also review by

Hall and Perona (2012)). Overall, these findings did not support that idea that this combined model would be a better model of ADHD, but they did continue to identify

ADHD-like effects in DAT +/- mice. These were further explored in the experiments presented in chapters 4, 5 and 6.

3.0 The DAT KO model of ADHD: Sex and the effects of partial DAT deletion

One outcome of the locomotor study in Chapter 3 was evidence of slight hyperactivity in male DAT +/- mice, but very little hyperactivity in female DAT +/- mice. This was observed again in the experiments presented in Chapter 5. In the locomotor study in chapter 4, a slight hyperactivity was observed in female DAT +/- mice, but not male DAT +/- mice. The reasons for these slight differences between studies are not clear, but there were differences in the testing conditions between the experiments, particularly the duration of locomotor testing and repeated exposure to testing conditions when drugs were being tested using within-subjects designs (Chapter 289

5). In the first study (Chapter 3), CAR was also slightly impaired in DAT +/- mice of both sexes in the initial study in Chapter 3, an effect that was reduced by social isolation early in life. Only very slight differences in CAR performance were observed in DAT +/- mice in the experiments in Chapters 4 and 5, but large deficits were observed in DAT -/- mice.

Although the initial sex effects on locomotor hyperactivity were different in the experiments presented in Chapters 3 and 4, there certainly was evidence for sex- dependent effects on DAT deletions throughout these studies in both DAT -/- and DAT

+/- mice. Thus, the PPI deficits in chapter 3 were only significant in female DAT -/- mice and not male DAT -/- mice. DAT +/- mice of either sex did not have PPI deficits. In the experiments presented in Chapters 3 and 4, CAR deficits in DAT -/- were much larger in male than in female DAT -/- mice.

The studies presented in Chapters 3, 4, and 5 replicated many effects previously observed in DAT -/- mice, and further demonstrated that the observation of these effects is partially sex-dependent, and that effects can be observed in DAT +/- mice under some circumstances. The final study presented in this dissertation explored attention and impulsivity using the 5-choice continuous performance task (5-CCPT). Female mice were not studied here because of testing limitations. Preliminary studies in DAT -/- mice found that they were so impaired that they could not even complete the most basic versions of this task – once they realized that food came from the food hopper at the back of the chamber, they spent all of their time near it, ignoring all other features of the chamber. This is worthy of further investigation, but investigations of DAT +/- mice were illuminating, ad provide additional evidence in support of the DAT +/- model as a model 290

of ADHD. There was limited evidence for impaired attention in this task; the primary impairments observed in DAT +/- mice were premature responses, indicative of motor impulsivity. An important aspect of the 5-CCPT is a go/no-go component that accentuates impulsive responses. Although previous findings in the CAR test (Yamashita et al., 2013), replicated several times in the current report, can be interpreted as an impulsive response, there are also other interpretations, such as impaired executive function. The findings of impulsivity in the 5-CCPT are unambiguous, and really confirm for the first time that DAT +/- mice are impulsive.

The findings discussed above indicate that the DAT KO model has face validity as a model of ADHD. and perhaps even more so the DAT +/- model based on the findings in the 5-CCPT. Perhaps of even more relevance for the validity of DAT +/- mice as a model of ADHD, as well as the DAT -/- model, is the ability of effective ADHD treatments to reverse the ADHD-like deficits discussed above. In Chapter 3, amphetamine was found to reduce locomotion in DAT +/- mice while it increased locomotion in DAT +/+ mice (e.g. the paradoxical response to locomotor stimulants that is characteristic of ADHD). A similar ability of atomoxetine to reduce locomotor activity of male DAT +/- mice was found in Chapter 5, even though this drug did not affect hyperactivity in male DAT -/- mice. In female mice atomoxetine reduced activity in mice of all 3 genotypes, although it was perhaps least effective in female DAT -/- mice. In the

CAR test (Chapter 5) atomoxetine improved CAR performance in DAT +/- and DAT -/- mice of both sexes. In chapter 6, amphetamine also reduced impulsivity in DAT +/- mice in the 5-CCPT.

291

In considering the potential of DAT +/- mice to model ADHD, an important consideration is the ability of drugs that treat ADHD to affect DAT +/- behavioral deficits. This was shown for several typical ADHD-like drugs, as well as for a novel therapeutic approach discussed below.

4.0 5-HT1B Antagonism as a potential treatment for ADHD

The idea that 5HT1B antagonism might reverse behavioral deficits in the DAT

KO model is based in part upon an initial finding that it reduces hyperlocomotion in DAT

-/- at a dose of 20 mg/kg IP (Hall, Sora, Hen, & Uhl, 2014). This was not found to be the case in the first locomotor study using this dose (Chapter 4), however SB 224289 did reduce the small deficits in PPI and CAR observed in female DAT -/- mice in those studies. A broader range of SB 224289 doses was examined in Chapter 5, and 5HT1B antagonism was found to reduce locomotion in male DAT +/- and DAT -/- mice. Similar trends were observed in females, but these effects were not significant. SB 224289 also improved CAR performance in male DAT +/- and DAT -/- mice, and in female DAT -/- mice (Chapter 5). These effects were not as robust as was observed for atomoxetine, which may either indicate that this mechanism is less important for the constructs underlying impaired CAR performance, as opposed to hyperactivity, or perhaps that the drug did not act for long enough (the test was 1 hr long). Finally, and quite importantly,

SB 224289 also reduced impulsivity in DAT +/- as assessed in the 5-CCPT, demonstrating its effectiveness in important cognitive symptoms that are observed both in the DAT +/- model and in ADHD.

292

5.0 Final Conclusions

In summary, these findings have provided additional support for the DAT -/- mouse model of ADHD, finding evidence of both face and predictive validity. At the same time, these studies have identified limitations of model, perhaps reflecting the consequences of such severe DAT perturbations in humans (Hansen et al., 2014).

Although DAT +/- mice did not demonstrate all of the deficits found in DAT -/- mice, many deficits were found, including impulsivity in the 5-CCPT, providing substantial evidence for the potential of the DAT +/- model as a model of ADHD. This face validity of the DAT +/- model was further supported by predictive validity. Many of the deficits in DAT -/- mice, as well as DAT +/- mice, were reversed or reduced by effective ADHD medications in these studies. Moreover, many of these deficits were also ameliorated by the 5HT1B antagonist SB 224289, suggesting that the mechanism may provide an alternative, non-stimulant approach to the treatment of ADHD.

293

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