Prenatal nicotine sex-dependently alters adolescent dopamine system development.

Despite persistent public health initiatives, many women continue to smoke during pregnancy. Since maternal smoking has been linked to persisting sex-dependent neurobehavioral deficits in offspring, some consider nicotine to be a safer alternative to tobacco during pregnancy, and the use of electronic nicotine delivery systems is on the rise. We presently show, however, that sustained exposure to low doses of nicotine during fetal development, approximating plasma levels seen clinically with the nicotine patch, produces substantial changes in developing corticostriatal dopamine systems in adolescence. Briefly, pregnant dams were implanted on gestational day 4 with an osmotic minipump that delivered either saline (GS) or nicotine (3 mg/kg/day) (GN) for two weeks. At birth, pups were cross-fostered with treatment naïve dams and were handled daily. Biochemical analyses, signaling assays, and behavioral responses to cocaine were assessed on postnatal day 32, representative of adolescence in the rodent. GN treatment had both sex-dependent and sex-independent effects on prefrontal dopamine systems, altering Catechol-O-methyl transferase (COMT)-dependent dopamine turnover in males and norepinephrine transporter (NET) binding expression in both sexes. GN enhanced cocaine-induced locomotor activity in females, concomitant with GN-induced reductions in striatal dopamine transporter (DAT) binding. GN enhanced ventral striatal D2-like receptor expression and G-protein coupling, while altering the roles of D2 and D3 receptors in cocaine-induced behaviors. These data show that low-dose prenatal nicotine treatment sex-dependently alters corticostriatal dopamine system development, which may underlie clinical deficits seen in adolescents exposed to tobacco or nicotine in utero

The impact of psychostimulant administration during development on adult brain functions controlling motivation, impulsivity and cognition.

ADHD pharmacotherapy uses methylphenidate (MPH), D-amphetamine (D- amph), two psychostimulants targeting dopamine transporters, or atomoxetine (ATX), specifically targeting norepinephrine transporters. We have assessed the pharmacological mechanisms of these three drugs on the in vitro efflux of neurotransmitters in rat prefrontal cortex (PFC) and striatal slices as well as on the in vivo electrical activities of PFC pyramidal neurons, striatal medium spiny neurons, ventral tegmental area dopamine neurons or dorsal raphe nucleus serotonin neurons, using single cell extracellular electrophysiological recording techniques. We have also tested whether chronic methylphenidate treatment, during either adolescence or adulthood, could have long-lasting consequences on body growth, depression and neuronal functions. Release experiments showed that all ADHD drugs induce dose-dependent dopamine efflux in both the PFC and striatum, with different efficacies, while only D- amph induced cortical norepinephrine efflux. Atomoxetine induced an unexpected massive dopamine outflow in striatal regions, by mechanisms that depend on physiological parameters. Our electrophysiological studies indicate that all three drugs equally stimulate the excitability of PFC pyramidal neurons, in basal and NMDA-evoked conditions, when administered acutely (3 mg/kg). While the electrophysiological effects elicited by psychostimulants may be dependent on D1 receptor activation, those induced by atomoxetine relied on different mechanisms. In the ventral tegmental area (VTA), methylphenidate (2 mg/kg), but not atomoxetine, induced firing and burst activity reductions, through dopamine D2 autoreceptor activation. Reversal of such effects (eticlopride 0.2 mg/kg) revealed an excitatory effect of methylphenidate on midbrain dopamine neurons that appear to be dependent on glutamate pathways and the combination of D1 and alpha-1 receptors. Finally, acute intraperitoneal psychostimulant injections increased vertical locomotor activity as well as NMDA2B protein expression in the striatum. Some animals chronically treated with intraperitoneal administrations (methylphenidate 4 mg/kg/day or saline 1.2 ml/kg/day) showed decreased body weight gain. Voluntary oral methylphenidate intake induces desensitisation to subsequent intravenous methylphenidate challenges, without altering dopamine D2 receptor plasticity. Significant decreases in striatal NMDA2B protein expression were observed in animals chronically treated. After adolescent MPH treatment, midbrain dopaminergic neurons do not display either desensitisation or sensitisation to intravenous methylphenidate re-challenges. However, partial dopamine D2 receptor desensitisation was observed in midbrain dopamine neurons. Using behavioural experiments, cross-sensitisation between adolescent methylphenidate exposure and later-life D-amphetamine challenge was observed. Significant decreases in striatal NMDA2B protein expression were observed in animals chronically treated, while striatal medium spiny neurons showed decreased sensitivities to locally applied NMDA and dopamine. While caffeine is devoid of action on baseline spike generation and burst activity of dopamine neurons, nicotine induces either firing rate enhancement, firing rate reduction, or has no consequences. Adolescent methylphenidate treatment leads to decreased neuronal sensitivities to the combination of nicotine, MPH and eticlopride, compared to controls. Finally, nicotine partially prevented D-amphetamine-induced increase of rearing activities. Our results show that increases in the excitability of PFC neurons in basal conditions and via NMDA receptor activation may be involved in the therapeutic response to ADHD drugs. Long-term consequences were observed after psychostimulant exposure. Such novel findings strengthen the mixed hypothesis in ADHD, whereby both dopamine and glutamate neurotransmissions are dysregulated. Therefore, ADHD therapy may now focus on adequate balancing between glutamate and dopamine

Developmental plasticity and circuit mechanisms of dopamine-modulated aggression

Aggression and violence pose a significant public health concern to society. Aggression is a highly conserved behavior that shares common biological correlates across species. While aggression developed as an evolutionary adaptation to competition, its untimely and uncontrolled expression is maladaptive and presents itself in a number of neuropsychiatric disorders. A mechanistic hypothesis for pathological aggression links aberrant behavior with heightened dopamine function. However, while dopamine hyper-activity is a neural correlate of aggression, the developmental aspects and circuit level contributions of dopaminergic signaling have not been elucidated. In this dissertation, I aim to address these questions regarding the specifics of dopamine function in a murine model of aggressive behavior. In chapter I, I provide a review of the literature that describes the current state of research on aggression. I describe the background elements that lay the foundation for experimental questions and original data presented in later chapters. I introduce, in detail, published studies that describe the clinical manifestation and epidemiological spread, the dominant categories, the anatomy and physiology, and the pharmacology of aggression, with a particular emphasis on the dopaminergic system. Finally, I describe instances of genetic and environmental risk factors impacting aggression, concluding with studies revealing an important role for interactions among genetics, environmental factors, and age in the development of aggression. In chapter II, I investigate the developmental origins of aggression by examining sensitive periods during which perturbations to the dopaminergic system impact adult aggressive behavior. Previous work in our laboratory has concluded that periadolescent (postnatal days 22-41) elevation in dopamine, via transient dopamine transporter blockade, leads to increased adult aggression and heightened response to amphetamine. I expanded on these findings by temporally refining the opening and closing of this window of sensitivity, specifically to postnatal days 32 to 41, during which increases in dopaminergic tone increase adult aggression and behavioral sensitivity to psychostimulants. The potentiated response to amphetamine indicated to us a state of altered dopaminergic physiology. We next validated this hypothesis and found increased firing rate (in vitro), and increased bursting and population activity (in vivo) at baseline. These data indicate that elevated periadolescent dopamine impacts maturation of the dopamine system, leading to a hyper-active dopaminergic and aggressive predisposition. In conclusion, this chapter introduces a developmental component to the hyper-dopaminergic model of aggression. In chapter III, I report a series of experiments exploring the direct and causal involvement of dopamine in driving aggression. While dopamine hyper-activity is a neural correlate of aggression, the precise brain circuits involved have not been elucidated. Using optogenetics, I established a causal role for the ventral tegmental area (a key source of dopamine) in aggression modulation. I further advanced this finding by demonstrating that the modulatory role of dopamine, is population- and projection-specific. I found that activity of ventral tegmental area, but not substantia nigra, dopamine neurons promotes aggression. Furthermore, controlled stimulation of ventral tegmental area dopaminergic terminals in the lateral septum, but not the nucleus accumbens, mediates increased aggression. I selectively traced connectivity between the lateral septum and the ventral tegmental area using a Cre-driven, population-specific viral vector. I used this virus to show that anatomically distinct clusters of ventral tegmental area dopamine cells send projections to the lateral septum and the nucleus accumbens, thereby dissociating the two target sites both behaviorally and anatomically. Furthermore, I found that while local dopamine release in the lateral septum increases aggression, it has no bearing on reward behaviors thus indicating a stronger association with impulsive, and not motivated, aggression. In conclusion, this chapter offers causal evidence for dopamine’s role in modulating impulsive aggression by identifying a distinct pathway from the ventral tegmental area to the lateral septum that controls aggression. In the work described in chapter IV, my aim was to determine the mechanism underlying ventral tegmental area to lateral septum dopamine-mediated aggression. I first characterized the expression of dopamine receptors in the lateral septum and found that D2 receptors heavily colocalize with the dominant population of neurons in the lateral septum, i.e. GABAergic cells. Moreover, the D2 receptors are perfectly aligned with incoming dopamine afferents. Next we investigated, in acute brain slices, how D2 signaling affects lateral septum function. We revealed that activating D2 receptors hyperpolarizes D2-expressing lateral septum neurons. This effect was abolished with bath application of the D2 receptor antagonist, sulpiride. We validated the functional involvement of post-synaptic D2 signaling in a behavioral test, and found that the aggression induced by direct terminal release of dopamine at the lateral septum is reversed by acutely blocking local D2 receptor signaling. In conclusion, this chapter demonstrates that the ventral tegmental area to lateral septum dopamine pathway, via D2-mediated inhibition of GABAergic lateral septum neurons, is necessary to drive ventral tegmental area-triggered aggression. In chapter V, I engage in a general discussion addressing how the findings from each chapter can be linked to provide a more comprehensive outlook on environmental and genetic risk factors that can modulate ventral tegmental area-triggered aggression. I discuss possible pre- and post-synaptic mechanisms that could impact the functionality of the identified dopaminergic ventral tegmental area to lateral septum pathway. Moreover, in distinguishing this specific dopamine circuit and lateral septum D2 signaling as an underlying correlate of violent pathology, this dissertation aims to evoke deeper understanding of the mechanism of current antipsychotics used to manage aggression. I end this dissertation by proposing new empirical questions, techniques and lines of research that could further develop my findings as well strengthen the links between dominant models of aggression that exist in the field today

METHYLPHENIDATE AND ATOMOXETINE TREATMENT DURING ADOLESCENCE IN THE SPONTANEOUSLY HYPERTENSIVE RAT: MECHANISMS UNDERLYING HIGH COCAINE ABUSE LIABILITY IN ATTENTION DEFICIT/HYPERACTIVITY DISORDER

Effects of pharmacotherapies for Attention Deficit/Hyperactivity Disorder (ADHD) on cocaine abuse liability in ADHD are not understood. Spontaneously Hypertensive Rats (SHR), an ADHD model, exhibited greater cocaine self-administration than control Wistar-Kyoto and Wistar rats. Methylphenidate, but not atomoxetine during adolescence enhanced cocaine self-administration in adult SHRs compared to controls. The mesocortical dopaminergic system, including medial prefrontal (mPFC) and orbitofrontal (OFC) cortices, is important for ADHD and cocaine addiction. Dopamine and norepinephrine transporter (DAT and NET) are molecular targets for methylphenidate, atomoxetine and cocaine action. In the current studies, SHR, Wistar-Kyoto and Wistar were administered methylphenidate (1.5 mg/kg/day, p.o.), atomoxetine (0.3 mg/kg/day, i.p.) or vehicle during adolescence (postnatal day 28-55). During adulthood (\u3e77 days), DAT and NET functions in mPFC and OFC were determined as neurochemical mechanisms and locomotor sensitization to cocaine, and impulsivity under differential reinforcement of low rates 30-second (DRL30) schedule were evaluated as behavioral mechanisms associated with greater cocaine self-administration in methylphenidate-treated SHRs. Maximal velocity of [3H]dopamine uptake (Vmax) by DAT and DAT cellular distribution in mPFC and OFC did not differ between vehicle-control, adult SHR, Wistar-Kyoto and Wistar. Methylphenidate increased DAT Vmax, but not cell-surface expression, in SHR mPFC. In contrast, atomoxetine decreased Vmax and cell-surface expression in SHR OFC. Compared to control strains, norepinephrine uptake by NET in the OFC was increased in vehicle-administered SHR; methylphenidate during adolescence normalized NET function in SHR OFC. Locomotor sensitization was greater in SHR compared to control, and was not altered by methylphenidate. Under DRL30, methylphenidate increased burst responses in adult SHR compared to vehicle control as well as methylphenidate-treated Wistar-Kyoto and Wistar, indicating increased impulsivity. Increased OFC NET function, increased impulsivity and cocaine sensitivity may be the neurobehavioral mechanisms associated with the increased cocaine self-administration in SHR. Increased mPFC DAT function may underlie the enhanced impulsivity and cocaine self-administration in SHR administered methylphenidate during adolescence. Decreased OFC DAT function from atomoxetine-treated SHR may explain the reduced cocaine self-administration relative to methylphenidate. Thus, methylphenidate during adolescence in ADHD may increase risk for cocaine abuse, while atomoxetine may represent a therapeutic alternative for at-risk adolescents with ADHD

Moderator Effects of Working Memory on Symptom Stability in Attention-Deficit/Hyperactivity Disorder by Dopamine D1 and D2 Receptor Polymorphisms During Development

Background: Developmental changes in dopaminergic pathways in the prefrontal cortex (PFC) that are important for working memory have been hypothesized to play a central role in the trajectory of attention-deficit/hyperactivity disorder (ADHD), but not the initial onset of the disorder. This dissertation research examines whether dopamine receptor D1 (DRD1) and dopamine receptor D2 (DRD2) gene polymorphisms moderate the association between improvements in working memory and declines in attention problems in ADHD from childhood to adolescence/young adulthood. Methods: Participants were 76 racially/ethnically diverse youth diagnosed with ADHD in childhood and followed prospectively for almost 10 years. Stability of ADHD symptomatology was measured as a quantitative trait using the Attention Problems scale from the Child Behavior Checklist collected both in childhood and adolescence/young adulthood. Digit Span Forward and Digit Span Backward were administered at both time points to assess working memory maintenance and manipulation, respectively. Genotype and age were moderator variables. Results: DRD1 and DRD2 polymorphisms were associated with the stability of attention problems in adolescence/young adulthood, but not childhood. DRD1 polymorphisms, but not DRD2, significantly moderated the association between working memory and attention problems, with the strongest effects evidenced during adolescence/young adulthood. Notably, DRD1 moderation of working memory on attention problems was specific to manipulation performance. Conclusions: Attention problems decreased over the course of almost 10 years if manipulation concomitantly improved during this period of development in a subgroup of individuals with childhood-diagnosed ADHD depending on their genetic makeup

The etiology of ADHD

Attention Deficit/Hyperactivity Disorder (“ADHD”) is a complex multi-factorial disorder that was first described in the late 1800s as a defect in moral control. By the early 1900s, ADHD shifted away from being a behavioral-based to a neurobiological-based disorder. During this period, individuals with ADHD were classified as having minimal brain damage. Early studies focused on the clinical presentation of ADHD. As advances in neuroimaging and molecular marker techniques started to develop, researchers were able to focus more on the neurobiological aspects of ADHD. This shift was instrumental to both the diagnosis and treatment of ADHD. This paper surveys the existing literature on ADHD in an attempt to elucidate its etiology. While several areas of research seem promising, so far, no single major contributor to ADHD has been identified. This paper first looks at the history behind ADHD. The historical background was instrumental in directing the course of ADHD research. Next, the Diagnostic and Statistical Manual of Mental Disorders (“DSM”) is examined with a focus on the changes made to DSM-IV and reflected in DSM-V. While DSM is a valuable diagnostic tool, its purpose in elucidating the etiology behind ADHD is questionable. Despite that, discussion of the DSM is necessary as it is impossible to study a disorder without delineating the normal from the abnormal. What follows this discussion is a brief overview of comorbidities that are often associated, and possibly share, a common etiology with ADHD. The paper then examines the theories promulgated by researchers as to the neurobiological basis of ADHD. This examination is followed by a discussion of recent findings into the pathology behind ADHD, which mainly centers around differences in brain structure and connectivity. Further analysis of these studies reveals that sex plays an instrumental role in the type of brain abnormalities found in ADHD children. In addition, delays in brain development are analyzed, and age is discussed as a factor in the presentation of ADHD. This paper goes on to examine genetics as a contributor to the etiology of ADHD. This examination proves fruitful as several genes of interest seem to indicate a hereditary component of ADHD. Finally, treatment options such as psychosocial therapy and medications that help ADHD patients maintain a quality of life, are discussed. By studying the mechanism of action underlying these medications, additional clues as to the etiology of ADHD may be discovered. There is still a long way to go before a complete picture of ADHD emerges. Already, studies are showing that race and environmental factors play a role in how ADHD presents. These two areas have rarely been studied and doing so will only serve to enhance the current understanding of ADHD. Despite an incomplete picture, the scientific community has come a long way from the 1800s where ADHD was thought to result from a defect in moral control. With early diagnosis and proper treatment, the ADHD individuals of today can live a life as close to that of their neurotypical peers as possible

PRECLINICAL EVALUATION OF LOBELINE FOR THE TREATMENT OF ADHD: COMPARISON WITH PSYCHOSTIMULANT THERAPIES

This dissertation work investigated the effect of acute and repeated in vivo administration of lobeline on dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) function. The effects of lobeline were then compared to the effects of acute and repeated in vivo administration of methylphenidate and amphetamine to determine if lobeline produced similar effects compared to these Attention Deficit Hyperactivity Disorder (ADHD) medications. These medications are considered the first line of pharmacotherapy for ADHD, although there is a growing concern associated with their potential for abuse and other side effects. This merits the need for novel ADHD treatments that have a safer side effect profile. If lobeline alters DAT and VMAT2 function in the same way as methylphenidate or amphetamine, further investigation may be necessary to evaluate lobeline as a potential treatment for ADHD. Kinetic analysis of [3H]dopamine (DA) was utilized to determine the effect on DAT and VMAT2 function in rat striatum. Results from the DAT experiments, revealed that lobeline as well as amphetamine had no effect on DAT function. However, methylphenidate increased DAT function after acute and 7-day treatment. None of the drug treatment regimens altered Km. To determine if the methylphenidateinduced increase in DAT function was due to DAT trafficking, biotinylation and Western blot analyses were performed. Acute administration of methylphenidate did not alter surface DAT, however repeated administration of methylphenidate for 7 days decreased intracellular DAT, suggesting that methylphenidate redistributes DAT in a time-dependent manner. Similar results were found in the VMAT2 experiments. Lobeline and amphetamine had no effect on VMAT2 function after acute or repeated administration. Amphetamine decreased the Km after repeated administration for 7 days. Methylphenidate increased VMAT2 function after acute and repeated administration for 7 days. The overall results of these experiments suggest that methylphenidate interacts with DAT and VMAT2 in a different manner than amphetamine and lobeline. In addition, since lobeline and amphetamine had no effect on DAT and VMAT2 function, further investigation is warranted to elucidate the underlying mechanisms of the therapeutic actions of these agents. This additional information will aid in the development of novel treatments for ADHD

Towards the convergent therapeutic potential of GPCRs in autism spectrum disorders

Changes in genetic and/or environmental factors to developing neural circuits and subsequent synaptic functions are known to be a causative underlying the varied socio-emotional behavioural patterns associated with autism spectrum disorders (ASD). Seven transmembrane G protein-coupled receptors (GPCRs) comprising the largest family of cell-surface receptors, mediate the transfer of extracellular signals to downstream cellular responses. Disruption of GPCR and their signalling have been implicated as a convergent pathologic mechanism of ASD. Here, we aim to review the literature about the 23 GPCRs that are genetically associated to ASD pathology according to Simons Foundation Autism Research Initiative (SFARI) database such as oxytocin (OXTR) and vasopressin (V1A, V1B) receptors, metabotropic glutamate (mGlu5, mGlu7) and gamma-aminobutyric acid (GABAB) receptors, dopamine (D1, D2), serotoninergic (5-HT1B and additionally included the 5-HT2A, 5-HT7 receptors for their strong relevance to ASD), adrenergic ($\beta$2) and cholinergic (M3) receptors, adenosine (A2A, A3) receptors, angiotensin (AT2) receptors, cannabinoid (CB1) receptors, chemokine (CX3CR1) receptors, orphan (GPR37, GPR85) and olfactory (OR1C1, OR2M4, OR2T10, OR52M1) receptors. We discussed the genetic variants, relation to core ASD behavioural deficits and update on pharmacological compounds targeting these 23 GPCRs. Of these OTR, V1A, mGlu5, D2, 5-HT2A, CB1, and GPR37 serve as the best therapeutic targets and have potential towards core domains of ASD pathology. With a functional crosstalk between different GPCRs and converging pharmacological responses, there is an urge to develop novel therapeutic strategies based on multiple GPCRs to reduce the socioeconomic burden associated with ASD and we strongly emphasize the need to prioritize the increased clinical trials targeting the multiple GPCRs

Neurobiological mechanisms of heterogeneous nuclear ribonucleoprotein H1 in methamphetamine stimulant and addictive behaviors

Addiction to psychostimulants such as methamphetamine (MA) is a significant public health issue in the United States with no FDA-approved pharmacological interventions. MA addiction is a heritable neuropsychiatric disorder, however, its genetic basis is almost entirely unknown. Available human genome-wide association studies (GWAS) lack sufficient power to detect the influence of common genetic variation on the risk of addiction. Mammalian model organisms offer an attractive alternative to more rapidly uncover novel genetic factors that contribute to addiction-relevant neurobehavioral traits. Using quantitative trait locus (QTL) mapping in mice, we identified a locus on chromosome 11 that contributed to a decrease in sensitivity to the locomotor stimulant properties of MA. To fine map this QTL, we generated interval-specific congenic lines and deduced a 206 kb critical interval on chromosome 11 that contained only two protein coding genes (Rufy1 and Hnrnph1). Replicate mouse lines heterozygous for Transcription Activator-like Effector Nucleases (TALENs)-induced frameshift deletions in Hnrnph1 (Hnrnph1+/-), but not in Rufy1 (Rufy1+/-), recapitulated the decrease in MA sensitivity observed in congenic mice; thus, identifying Hnrnph1 as a novel quantitative trait gene for MA sensitivity. Hnrnph1, an RNA-binding protein, has not previously been identified in human GWAS of neuropsychiatric disorders but has been implicated in mu-opioid receptor splicing associated with heroin dependence. The primary objectives of this dissertation is to (1) detail the forward genetic and reverse genetic approaches taken to identify Hnrnph1 as a quantitative trait gene for MA sensitivity; (2) assess the MA addiction-relevant behaviors presented by Hnrnph1+/- mice through conditioned place preference (CPP) and oral self-administration procedures; and (3) identify the neurobiological mechanisms through which Hnrnph1 affects behavior via transcriptome, immunohistochemical and neurochemical assessments of the mesocorticolimbic dopamine circuit. Overall, Hnrnph1+/- mice display increased dopaminergic innervation and MA dose-dependent dopamine release in nucleus accumbens, which could underlie reduced drug sensitivity, reward, and reinforcement. The results of this thesis provide substantial evidence to implicate Hnrnph1 in MA addiction