Our lab has created a construct valid mouse model based on the dopamine transporter (DAT) Val559 coding variant to elucidate neurobiological mechanisms supporting ADHD and other dopamine (DA)-linked comorbid disorders. Previously we demonstrated that the DAT Val559 mice have altered behavioral response to amphetamine, postulating that it was through a constitutively active D2-DAT interaction that resulted in reduced vesicular release. We have now further explored this altered drug response by examining downstream signaling proteins that are known to play a role in amphetamine induced locomotor behavior. Additionally, we sought to extend our analysis to understand the effects of life long expression of this variant on behavior associated with clinically-relevant phenotypes. We utilized the 5-choice serial reaction time task to probe for evidence of alterations in cognitive, attention, and impulsivity domains. We observed a schedule-dependent impulsivity in the DAT Val559 mice relative to WT controls, and demonstrated that the DAT Val559 mice specifically express a waiting impulsivity and not a deficit in motor inhibition as assessed by the Go/NoGo paradigm. Additionally, we determined that the waiting impulsivity was driven by an enhanced motivational state, rather than an alteration in the ability of the DAT Val559 mice to time intervals. Finally, DAT Val559 mice were demonstrated to be more susceptible to the formation of a dysregulated perseverative checking behavior under a devaluated state across goal-directed and habit contexts. Ultimately the DAT Val599 mouse represents a salient opportunity to understand perturbed behaviors seen in neuropsychiatric conditions associated with DAergic dysfunction.

Contributions of Metallo-Beta-Lactamase Domain Containing Protein 1 (MBLAC1) to the Neurobiological Actions of Ceftriaxone Cassandra Lynn Retzlaff <p> Dissertation under the direction of Professor Randy Blakely, PhD <p> Recently identified glial-expressed C. elegans gene, swip-10, encodes a metallo-beta-lactamase domain-containing protein, which limits glutamate-dependent changes in dopamine neuron excitability. Bioinformatic analyses identified MBLAC1 as the likely mammalian orthologue of swip-10. Ceftriaxone, a beta-lactam antibiotic, has been reported to act independently of its antimicrobial actions to normalize perturbed central nervous system glutamate levels, principally by elevating expression of glial glutamate transporters. Housing a canonical beta-lactam binding domain, MBLAC1, stood out as a possible molecular target for ceftriaxone, of which one is currently unknown. Using multiple approaches, evidence presented asserts the specific, high affinity binding of ceftriaxone to MBLAC1. Furthermore, the creation of a novel knockout mouse model was utilized in order to test these hypotheses in vivo, and to establish a role for MBLAC1 in the brain.

Includes bibliographical references. / Background: Social anxiety disorder (SAD) is a common psychiatric condition characterised by fear and avoidance of social situations. Lifetime prevalence is 5-16% and co-morbidity with other mood and substance abuse disorders is common. Symptoms including cognitive, behavioural and physiological components vary between individuals. Of these, blushing and gaze fear and avoidance are regarded as cardinal symptoms. First line treatment of SAD involves SSRIs and cognitive behavioural therapy, while surgery may also be considered for excessive blushing. Blushing and gaze avoidance are thought to have an evolutionary adaptive advantage, promoting the display of submissive behaviour and appeasement in threatening situations. MRI research has demonstrated differences on functional and structural neuroimaging between patients with SAD and healthy controls (HCs). However, little is known about the neurocircuitry underlying gaze fear and avoidance or increased blushing propensity or how the severity of these traits correlate with the neuroimaging differences found in SAD. In this research, I explored the neuroanatomy of blushing propensity and gaze fear and avoidance in the context of SAD. Methods: 18 SAD patients and 18 HCs underwent structural MRI scans and self-report scales were administered to assess their symptom severity, blushing propensity and gaze fear and avoidance. Structural data was analysed using voxel-based morphometry (VBM). Regression and contrast analyses were used to correlate blushing propensity and gaze anxiety and avoidance symptoms with brain volumes, controlling for total grey matter volume, age and level of education. Results: Anxiety, blushing propensity and gaze fear and avoidance symptoms were all significantly higher in SAD patients (p<0.001). Brainstem volumes were increased for higher blushing scores a (p<0.01), while the volumes of left inferior parietal lobe b (p=0.04) and left occipital cortex a (p<0.01) were decreased. With increased gaze fear and avoidance, there were associated decreases in the right posterior cingulate cortex a (p<0.01), right occipital lobe b (p=0.03) and right fusiform gyrus a (p<0.01). Increased blushing and gaze symptom severity considered together, was associated with increased brainstem volume a (p<0.01) and decreased pons/cerebellum b (p=0.001), right cerebellum b (p=0.009), left cerebellum c (p<0.001) and left inferior parietal lobe a (p<0.1), volumes. Contrast analysis of SAD and HC brain volumes revealed a greater grey matter volume in HCs in the regions of left occipital cortex (p<0.01), left anterior cingulate (p<0.01) and right inferior parietal lobe (p<0.01) when compared to SAD patients. Increased symptom severity in SAD was significantly associated with higher volumes in the left premotor cortex (p<0.01), right hippocampus (p<0.01), left orbitofrontal cortex (p<0.01) and right superior temporal cortex (p<0.01). Possible areas for of interest for volume differences between SAD and HCs include total grey matter volume (d =0.83), left and right anterior cingulate cortex (d =0.68 and d =0.65), and left and right dorsolateral prefrontal cortex (d =0.55 and d =0.54), yet these differences were not significantly different. (a uncorrected peak levels b uncorrected cluster level, c corrected cluster level). Conclusion: Differences in brain volumes pertaining to blushing and gaze fear and avoidance in SAD patients may be a contributing factor or a consequence of these core symptoms, and a potential biomarker for SAD. Future studies could build on this preliminary research with increased sample sizes, and determine the possible effects of reduced symptom severity and treatment options on brain structure and function. Most importantly, an investigation of the genetic underpinnings and functional neural correlates of blushing and gaze avoidance behaviour may enhance our understanding of the complex aetiology of these cardinal SAD symptoms, thereby improving our understanding of SAD as a psychiatric disorder and facilitating better patient care and management.

Certain high-risk communities in the Western Cape Province of South Africa where heavy maternal prenatal alcohol consumption is perpetuated by historical and societal challenges, have some of the highest prevalence rates of fetal alcohol syndrome (FAS) in the world. FAS has lifelong behavioural and cognitive consequences. Neuroimaging research aims to link deficits in brain structure and function to behavioural outcomes. Manual tracing is considered the gold standard of neuroanatomical volumetric analysis. Combined with neurobehavioural testing it can provide links between structure and function, but is time consuming and labour intensive. Automated segmentation programmes, such as FreeSurfer, are a faster alternative. The challenge is creating automated programmes that can provide results that are comparable to manual tracing, especially in a clinical sample. The aims of this thesis were to investigate (1) the effects of prenatal alcohol exposure (PAE) on the sizes of the caudate nucleus, nucleus accumbens, hippocampus and corpus callosum (CC) and potential relations of regional volumes with IQ and verbal learning, (2) to compare the performance of manual and automated segmentation methods in identifying alcohol-related changes in brain morphometry, and (3) to examine the effects of PAE on inter-hemispheric transfer during adolescence and potential relations of CC size with inter-hemispheric transfer deficits. Participants for this project were recruited from the Cape Town Longitudinal Cohort for whom alcohol exposure data were gathered prospectively from the mothers during pregnancy using the timeline follow-back approach. Participants had been diagnosed previously by two expert dysmorphologists as either control, non-syndromal heavily exposed (HE), partial FAS (PFAS) or FAS. High-resolution T1-weighted images were acquired using a sequence optimized for morphometric neuroanatomical analysis on a Siemens 3T Allegra MRI scanner for 71 right-handed children (9 FAS, 19 PFAS, 24 HE and 19 non-exposed controls) from this cohort at ages 9-11 years. Bilateral caudate nuclei, nucleus accumbens and hippocampi and the CC were manually traced using Multitracer. FreeSurfer was used for automated segmentation. All structures were segmented with both FreeSurfer versions 5.1 and 6.0 to compare progress within development of automated segmentation algorithms. Associations of volumes from manual tracing with IQ and performance on the California Verbal Learning Test-Children’s Version (CVLT-C) were also examined. Inter-hemispheric transfer was assessed using a finger localization task (FLT) administered to 74 participants (12 FAS, 16 PFAS, 14 HE, and 32 controls) from the same cohort at ages 16-17 years. Of these, 34 participants had completed MRI at 9-11 years. Higher levels of PAE were associated with reductions in CC area, as well as bilateral volume reductions in caudate nuclei and hippocampi, effects that remained significant after controlling for alcohol-related reductions in TIV (total intracranial volume). Amongst dysmorphic children (FAS/PFAS), poorer performance on the CVLT-C was related to larger hippocampi and smaller CC. Smaller CC was also associated with lower IQ and partially mediated the effect of PAE on IQ. Manual and automated comparisons showed good agreement in the caudate nuclei, which are simpler to segment, moderate to good agreement in the smaller, more complex nucleus accumbens and hippocampi, and poor agreement in the CC. The latter is not surprising, however, in view of the fact that manual tracing measured the average area of the CC on a mid-sagittal slice, while FreeSurfer measures CC volume over a number of contiguous slices. After controlling for confounders and adjustment for smaller TIV, the latest FreeSurfer version 6.0 provided evidence of alcohol-related volumetric brain reductions comparable to manual segmentation. Only the most severely affected children with FAS demonstrated inter-hemispheric transfer deficits, with the number of transfer-related errors tending to increase with decreasing CC volume among children with PAE. This study confirms and extends evidence of PAE-related decreases in subcortical and CC size and that callosal volume partially mediates alcohol-related impairment in IQ. Although FreeSurfer v 6.0 achieves automated segmentations that are comparable to manual tracing, even in a paediatric clinical sample, performance is more reliable in some structures than others. Improvement and standardization of CC segmentation is especially important given the vulnerability of the CC and its critical role in domains affected by PAE, including verbal learning, IQ and inter-hemispheric transfer of information.

Background: In recent years, methamphetamine use disorder (MUD), which is associated with adverse outcomes and represents a significant public health burden, has become highly prevalent in Cape Town, South Africa. Protracted methamphetamine (MA) use has been linked with neural dysfunction and working memory deficits. Although current treatments have shown limited efficacy in addressing MUD, recent evidence indicates the potential of utilizing tailored brief cognitive therapy programs and working memory training to improve outcomes. The current study aims to investigate the potential impact of brief cognitive therapy and using working memory training as an adjunct in the treatment of MUD. Methods: Participants were recruited from an in-patient drug rehabilitation centre in Cape Town. The sample (n = 26) consists of male patients (between the ages of 18–50) diagnosed with MUD. MUD patients were randomly split into 2 groups that received 4 weeks of treatment, i.e. treatment as usual (cognitive therapy only (NT) (n= 12)) and cognitive therapy with working memory training (CT) (n = 14). Neuroimaging and psychological data were collected from participants pre- and post- intervention to assess the relative impact of said interventions. Results: Behavioural outcome measures and the n-back working memory task adapted for fMRI were measured and compared pre- and post- intervention. No significant differences were present between groups prior treatment on behavioural measures, demographic measures, and fMRI activity. The brief cognitive therapy appeared to reduce depression and impulsivity scores over the course of the intervention, with scores slightly lower in the CT group. An FDR corrected whole-brain repeated measures ANOVA on the main effect of group indicated significant activation in the left posterior cingulate, left anterior cingulate, and left lingual gyrus. Post hoc t-tests were then conducted to follow up the group main effect and significant differences under FDR correction were observed in the NT group (in contrast to the CT group) indicating significantly more activity in the left superior temporal gyrus, left insula, right posterior declive, and right lingual gyrus. Significant differences were also observed under FDR correction on a posthoc test on the CT group (in contrast to the NT group) indicating significantly less activity in the left lingual gyrus, left posterior declive, and right cuneus. 5 Conclusions: The findings tentatively suggest that the working memory training adjunct may have slightly enhanced working memory maintenance brain function relative to the treatment as usual group post-intervention. The evidence also suggests that there may have been inefficient neural functioning in the treatment as usual group during the working memory task compared to the group receiving the working memory training adjunct. The results demonstrated that brief cognitive therapy treatment did somewhat reduce depressive symptoms and impulsivity in this study, with indications of subtle treatment gains in the cognitive training group. Overall, the current study (despite numerous limitations) provides preliminary and tentative evidence of the possible benefits of brief term cognitive therapy and the potential promise of using working memory training as a treatment adjunct.

Introduction: It has long been established that the Human Immunodeficiency Virus (HIV) and its effects, such as its impact on the immune system and then the numerous consequences on other biological systems including the central nervous system (CNS), have had a significant effect worldwide. This is particularly relevant in South Africa, where the prevalence of adults living with HIV remains high. However, with the improved access to antiretroviral therapy (ART), more children are now being born uninfected with HIV while still being exposed to the virus in utero. Exposure to HIV in utero may still negatively affect the developing brain of these children. However, the biological mechanisms involved in the neurodevelopmental outcomes in HIV-exposed uninfected (HEU) children are still largely unknown. Evidence from clinical studies showed that HEU children have an altered immune regulation compared to their unexposed counterparts, and this is hypothesised to play a role in neurodevelopmental outcomes in HEU children. The aim of this study was to evaluate the longitudinal relationship between the inflammatory environment of pregnant mothers living with HIV on ART and their children and the association of the inflammatory environment with neurodevelopmental outcomes in HEU children. Methods: This study was performed in a sub-sample of the Drakenstein Child Health Study (DCHS), a South African birth cohort of 1137 mother-infant pairs. This sub-study included mothers at ≈26 weeks gestation (n=267), their infants at 6-10 weeks (n=222) and children at 24-28 months (n=267). Maternal HIV status was determined at ≈26 weeks gestation. This sub-study included n=190 HIV negative mother-infant pairs and n=77 HIV positive mother-infant pairs. Serum inflammatory markers (Granulocyte-macrophage colony-stimulating factor (GM CSF), Interferon-γ (IFN-γ), Interleukin IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, tumour necrosis factor-α (TNF-α), neutrophil gelatinase-associated lipocalin (NGAL/Lcn2) and metalloproteinase-9 (MMP-9)) were analyzed in all study participants with a multiplex bead array and ELISA. The Bayley Scales of Infant and Toddler Development (Bayley-III) were used to assess the neurodevelopmental domains: cognitive, motor, language, social-emotional behaviour and adaptive behaviour at 24-28 months of age. Results: Mothers living with HIV on ART had significantly lower levels of the inflammatory markers GM-CSF and MMP9 compared to mothers without HIV. Serum levels of inflammatory markers IFN-γ and IL-1β were significantly lower in HEU infants at 6-10 weeks compared to HIV-unexposed uninfected (HUU) infants. At 24-28 months of age, HEU children also proved to have significantly lower serum levels of the inflammatory markers IFN-γ, IL-1β, IL-2 and IL-4 compared with HUU children. Increased levels of the inflammatory markers; GM-CSF, IFN-γ, IL-10, IL-12p70, IL-1β, IL-2, IL-4, IL-6 and Lcn2 in HEU infants at 6-10 weeks of age was associated with impaired motor neurodevelopment at 24-28 months of age. Conclusion: This is the first study to evaluate the longitudinal associations of immune markers with neurodevelopment in HEU children. The results show that maternal HIV infection was associated with lower levels of inflammatory markers in mothers and their children. Our results further indicate that an altered immune system in HEU infants, specifically at the earliest stages of life, was associated with impaired motor function at 2 years of age. These findings may provide further insights into the involvement of immune regulation, linking maternal HIV status and neurodevelopment in South African HEU children.

Epilepsy is more frequent in sub-Saharan Africa than the rest of the world due to high levels of brain infections by larvae of the pig cestode Taenia solium, a condition termed neurocysticercosis. Despite the large nature of the problem, little is known about how neurocysticercosis modulates neuronal responses to result in the development of seizures. In this thesis I have used the cestode Taenia crassiceps to develop multiple in vitro and in vivo models of neurocysticercosis in rodents. Utilising patch-clamp electrophysiology in organotypic hippocampal brain slices and chronic, wireless electrocorticographic recordings in freely moving animals I have explored how cestode larvae affect neuronal excitability in the brain across a range of time scales. First I demonstrate that homogenate of Taenia crassiceps larvae has a strong, acute excitatory effect on neurons, which is sufficient to trigger seizurelike events. The excitatory component of the homogenate was found to strongly activate glutamate receptors and not acetylcholine receptors nor acid-sensing ion channels. An enzymatic assay showed that the larval homogenate contains high levels of glutamate, explaining its acute excitatory effects on neurons. In the second part of my thesis I demonstrate that longer-term incubation of Taenia crassiceps homogenate with organotypic brain slices over the course of a day does not affect the intrinsic properties of pyramidal neurons nor the excitability of the neuronal network. In the final part of my thesis I established an in vivo model of neurocysticercosis. I found that intradermal inoculation together with multiple intracerebral injections of Taenia crassiceps homogenate did not result in the development of seizures over 3 months of chronic electrocorticography recordings. In addition, the seizure-threshold to picrotoxin, an excitotoxin, was not altered by Taenia crassiceps homogenate injection. Immunohistological analysis of the tissue below the injection site revealed no difference in astrocytes nor the number of microglia. However, microglial processes were observed to be retracted in the Taenia crassiceps group reflecting a moderate neuroinflammatory response. Together the data in my thesis provides novel insight into the acute and chronic effects of Taenia crassiceps homogenate on the excitability of neuronal networks with relevance to our understanding of neurocysticercosis.

Introduction and aims More than 90% of children with Autism Spectrum Disorder (ASD) live in low- and middle-income countries (LMIC) where there is a great need for culturally appropriate, scalable and effective early identification and intervention tools. Smartphone technology and application (‘apps’) may potentially play an important role in this regard. The Autism&Beyond iPhone App was designed as a potential screening tool for ASD risk in children aged 12-72 months. Here we investigated the technical feasibility and cultural acceptability of a smartphone app to determine risk for ASD in children aged 12-72 months in a naturalistic, low-income South African community setting. Methodology 37 typically-developing African children and their parents/carers were recruited from community centres in Khayelitsha Township, Cape Town, South Africa. We implemented a mixed-methods design, collecting both quantitative and qualitative data from participants in 2 stages. In stage 1, we collected quantitative data. With appropriate ethics and consent, parents completed a short technology questionnaire about their familiarity with and access to smartphones, internet and apps, followed by electronic iPhone-based demographic and ASD-related questionnaires. Next, children were shown 3 short videos of 30s each and a mirror stimulus on a study smartphone. The smartphone front facing (“selfie”) camera recorded video of the child’s facial expressions and head movement. Automated computer algorithms quantified positive emotions and time attending to stimuli. We validated the automatic coding by a) comparing the computer-generated analysis to human coding of facial expressions in a random sample (N=9), and b) comparing automated analysis of the South African data (N=33) with a matched American sample (N=33). In stage 2, a subset of families were invited to participate in focus group discussions to provide qualitative data on accessibility, acceptability, and cultural appropriateness of the app in their local community. Results Most parents (64%) owned a smartphone of which all (100%) were Android based, and many used Apps (45%). Human-automated coding showed excellent correlation for positive emotion (ICC= 0.95, 95% CI 0.81-0.99) and no statistically significant differences were observed between the South African and American sample in % time attending to the video stimuli. South African children, however, smiled less at the Toys&Rhymes (SA mean (SD) = 14% (24); USA mean (SD) = 31% (34); p=0.05) and Bunny video (SA mean (SD) = 12% (17); USA mean (SD) = 30% (0.27); p=0.006). Analysis of focus group data indicated that parents/carers found the App relatively easy to use, and would recommend it to others in their community provided the App and data transfer were free. Conclusion The results from this pilot study suggested the App to be technically accurate, accessible and culturally acceptable to families from a low-resource environment in South Africa. Given the differences in positive emotional response between the groups, careful consideration should be given to identify suitable stimuli if % time smiling is to be used as a global marker for autism risk across cultures and environments.

Tuberous Sclerosis Complex (TSC) is associated with many learning, behavioural, neurodevelopmental and psychiatric difficulties. Over 90% of individuals with TSC will have some of these concerns yet no more than 20% receive support and treatment, even though these issues may cause the greatest burden of disease in TSC. The Neuropsychiatry Panel at the 2012 TSC Consensus Conference coined the term TAND (TSC-Associated Neuropsychiatric Disorders) to capture the multidimensional concerns seen in TSC, and recommended that each person with TSC should be screened for TAND every year. To facilitate the process, a TAND Checklist was designed. Many professionals and families feel overwhelmed by the complexity of TAND and say that they do not know where to start and how to access relevant information, tips or 'next step' approaches. This may in part be due to the multi-dimensionality of TAND, and in part due to lack of access to clear, useful and evidence-based resources for TAND. This project aimed to address the complexity of TAND. The hypothesis was that, even though each individual will typically have their own unique TAND profile, there will be key natural TAND Clusters - combinations of behaviours across multi-dimensional levels - that will simplify further evaluations and treatment. The study was performed over 36 months, in two phases using a mixed-methods approach. Phase I was a pilot phase. TAND Checklist data were collected from 56 individuals with TSC in South Africa (n=20) and in Australia (n= 36). Using R, these data were explored with various multivariate data analysis techniques to identify suitable analysis methods for the identification of potential natural TAND clusters. WARD's cluster analysis method rendered six TAND clusters with good face validity, and convergence with a six-factor exploratory factor analysis solution. Pilot results suggested that a combination of cluster analysis and exploratory factor analysis methods may be able to identify clinically-meaningful natural TAND clusters. Phase II set out to replicate and expand on pilot results. TAND checklist data were collected from n=453 across six international TSC sites, and the multivariate analysis techniques identified in phase I were applied. WARD's method rendered seven natural TAND clusters with good clinical face validity. This data-driven strategy identified a 'Scholastic' cluster of TAND manifestations, a 'Neuropsychological' cluster, a 'Mood/Anxiety' cluster, an 'ASD-like' cluster, a 'Behaviours that Challenge' cluster, a 'Hyperactive/Impulsive' cluster, and an 'Eating/Sleeping' cluster. Results showed significant convergence with an exploratory factor analysis solution. The larger-scale study findings were remarkably consistent with pilot findings, supporting the robustness of these naturally occurring clusters. We propose that the seven natural TAND clusters identified can in future be used to generate clinical toolkits for use in real-life setting. In addition, findings suggest that the aetiology and molecular treatments of TAND may also show differential clustering across human and animal models, pointing towards novel hypotheses regarding neuropsychiatric phenomena in TSC to be explored in future studies.

Includes bibliographical references / While a number of studies have explored the functional neuroanatomy of social anxiety disorder (SAD), comparatively few studies have investigated the structural underpinnings in SAD. 18 psychopharmacologically and psychotherapeutically naïve adult patients with a primary Axis I diagnosis of generalized social anxiety disorder and 18 demographically (age, gender and education) matched healthy controls underwent 3T structural magnetic resonance imaging. A manual tracing protocol was specifically developed to compute the volume of the most prominent subcortical gray matter structures implicated in SAD by previous functional research. Cortical thickness was estimated using an automated algorithm and whole brain analyses of white matter structure were performed using FSL's tract - based spatial statistics comparing fractional anisotropy (FA), mean diffusivity (MD) in individuals with SAD. Manual tracing demonstrated that compared to controls, SAD patients showed an enlarged right globus pallidus. Cortical thickness analyses demonstrated significant cortical thinning in the left isthmus of the cingulate gyrus, the left temporal pole, and the left superior temporal gyrus. Analyses of white matter tractographic data demonstrated reduced FA in in the genu, splenium and tapetum of the corpus callosum. Additionally reduced FA was noticed in the fornix and the right cingulum. Reduced FA was also noted in bilateral corticospinal tracts and the right corona radiata. The results demonstrate structural alterations in limbic circuitry as well as involvement of the basal glanglia and their cortical projections and input pathways.