Schizophrenia is a mental illness with early adult onset. Prophylactic treatments would be clinically important and therefore we investigated the effect of two interventions: influenza vaccination of pregnant mothers and valproate treatment during late adolescence. Maternal immune response during pregnancy is thought to adversely affect brain development. We sought to assess whether immune activation by influenza vaccine could itself cause behavioural abnormalities in a mouse model. Our data suggest that further work is needed to make firm conclusions about the behavioural effects of the influenza vaccine. The second part of this thesis describes an analysis of valproate treatment on cortical neuron morphology in Disc1 L100P mice, a model for schizophrenia. Valproate was previously shown to prevent the onset of abnormal behaviours in Disc1 L100P mice. Contrary to expectations, valproate decreased apical spine density and the number of dendritic processes rather than reversing the dendritic deficits seen in Disc1 L100P mice.

Schizophrenia is a mental illness with early adult onset. Prophylactic treatments would be clinically important and therefore we investigated the effect of two interventions: influenza vaccination of pregnant mothers and valproate treatment during late adolescence. Maternal immune response during pregnancy is thought to adversely affect brain development. We sought to assess whether immune activation by influenza vaccine could itself cause behavioural abnormalities in a mouse model. Our data suggest that further work is needed to make firm conclusions about the behavioural effects of the influenza vaccine. The second part of this thesis describes an analysis of valproate treatment on cortical neuron morphology in Disc1 L100P mice, a model for schizophrenia. Valproate was previously shown to prevent the onset of abnormal behaviours in Disc1 L100P mice. Contrary to expectations, valproate decreased apical spine density and the number of dendritic processes rather than reversing the dendritic deficits seen in Disc1 L100P mice.

This research delves into idiopathic autism spectrum disorder (ASD), investigating the role of TrkB signaling pathways and BDNF regulation in the cortex. Additionally, it explores offering insights into maternal influences on mouse models. / Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by challenges in social interactions and repetitive behaviors. Prevalence of ASD is estimated to be 1 in 54 globally and is rising recently in many countries including Canada. ASD affects individuals differently, making diagnosis challenging. At present, no molecular diagnosis of ASD is available. Further, available medications only manage some symptoms of the disease and have adverse side effects in children. Therefore, there is a need for accurate molecular diagnostic tools to aid in molecular detection and treatment of ASD. To this end, a better understanding of the underlying molecular mechanisms that link ASD etiology to ASD-related behavior is crucial. While genetic factors contribute to syndromic ASD, most cases of ASD are idiopathic with unknown causes, influenced by a combination of epigenetic and environmental factors. TrkB and its downstream signaling pathways, such as Akt and Erk, are hyper-activated in syndromic ASD and hypo-activated in idiopathic cases. Therefore, drugs like rapamycin that inhibit the mTOR pathway downstream of TrkB are beneficial for syndromic ASD but not idiopathic cases. Additionally, insulin-like growth factor 1 (IGF-1), which mitigates ASD-related synaptic disruptions via Akt and Erk signaling, shows unchanged mRNA and protein levels along with its receptor in the idiopathic ASD fusiform gyrus. In ASD with either genetic or epigenetic/environmental causes, disruptions in synaptic connectivity are observed. Synaptic function is regulated by signaling pathways involving brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB), as well as their downstream signaling cascades such as MAPK and Akt. The existing literature suggests that there is an association between BDNF and TrkB signaling pathways and ASD. However, a serious gap in knowledge about the precise molecular role of TrkB in ASD pathology is that our current understanding is correlational in nature and based on observational studies that lack causal experiments. This underscores the importance of further research to understand the causative role of TrkB and its related molecular events in idiopathic ASD. The present work aims to provide a deeper understanding about the causative role of molecular mechanisms underlying TrkB signaling in ASD. ASD mouse models exhibit behaviors and molecular features resembling those observed in human ASD. Therefore, these mouse models are helpful tools for studying ASD. However, understudied physiological confounding factors, such as maternal age and parity, can introduce biases and add to data variability, thus negatively impacting the reproducibility and translational value of ASD mouse models. To achieve a reliable mouse model of ASD, we conducted our first study that examines the impact of maternal age and parity on pregnancy complications, neurodevelopment, and social behavior in mice. Results demonstrate that older maternal age and prior motherhood interact to ensure a normal, steady developmental rate and provide protective effects against anxiety, social impairment, and olfactory deficits. Given the current lack of clarity regarding the causative impact of TrkB on ASD pathology, our subsequent investigation sought to establish a causal relationship between TrkB signaling and ASD. We used the TrkB agonist, LM22A-4 treatment in a validated ASD mouse model. Our results demonstrate that treatment with LM22A-4 effectively rescues the core symptoms associated with ASD (social impairment and repetitive behavior). These findings indicate that impaired TrkB signaling is responsible for ASD-like behavior of valproic acid (VPA)-exposed mice. However, unlike TrkB-related molecular events occurring in the fusiform gyrus of idiopathic ASD, TrkB isoform protein levels, BDNF species, Akt, and Erk total protein levels and activation remained unchanged in VPA-exposed cortices compared to healthy control mice. Since our VPA mouse model does not replicate human idiopathic ASD, our study cannot draw a conclusion on how disruptions in these signaling pathways may contribute to the development and manifestation of ASD symptoms. Cortex is responsible for various aspects of social behavior that are impaired in ASD. However, regulatory mechanisms that are involved in ASD upstream of cortical TrkB and BDNF are not well known. BDNF expression is highly cell-and tissue-specific and is regulated by different sets of transcription factors in specific tissues. While NURR1, the BDNF regulator in midbrain neurons, is associated with ASD pathology, its specific role in regulation of cortical BDNF is not yet well-established. Our third study aimed to understand the role of NURR1 in regulating BDNF specifically in the cortex. We showed that in resting and depolarized neurons, when NURR1 is knocked down, BDNF mRNA levels remained unchanged, suggesting that NURR1 does not regulate BDNF in cortical neurons and highlighting the tissue-specificity of BDNF regulation. In summary, we address the understudied effects of maternal factors on mouse models, which enhances the reliability of ASD research. Further, our studies significantly enhance the understanding of ASD by elucidating the role of TrkB and its downstream signaling pathways in the behavioral aspects of the disorder. We also contribute to the knowledge of BDNF regulation in the cortex, a brain tissue with crucial roles in various aspects of social behavior. In a forward-looking approach, the results of our studies provide valuable insights into mouse modeling of idiopathic ASD and the potential role of TrkB in ASD behavioral symptoms. / Thesis / Candidate in Philosophy / Autism spectrum disorder (ASD) is a condition that is accompanied by challenges in social interaction and repetitive behaviors. ASD is a complicated condition because we do not fully understand all the details of how it works in the body. Studying ASD is important as it is the most challenging condition in children and it is becoming more common, especially in the last two decades. While scientists are developing molecular tools to improve ASD diagnosis and understand its biology, these tools are not widely used in clinics for ASD diagnosis yet. Also, the approved medications available can only help with managing some of the behavioral symptoms like self-harming behavior. Despite the pressing need to find a solution, our recent advancements have not yet brought us closer to a cure for ASD, mainly because of the complexity of the disorder. Therefore, identifying the specific ASD-related mechanisms at the molecular level that contribute to ASD-related behaviors is crucial for gaining a deeper understanding of the disease. In ASD, there are problems with how brain cells communicate with each other. This communication is controlled by certain molecules in the brain, such as brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB), along with other molecules. There is evidence suggesting a link between these molecules and ASD, but we have not fully understood their precise roles because most of the current knowledge is based on observations and correlations, rather than on establishing cause-and-effect relationships. To bridge this gap, our research focused on understanding TrkB's role in ASD. We required reliable mouse models. Since we aimed to induce ASD-like behaviors in mice using an ASD-causing chemical, it was crucial to ensure they were healthy beforehand. We needed to confirm that any social deficits or repetitive behaviors were not due to other factors, such as adverse infancy experiences or impaired interactions between mother and infant. We discovered that sexually mature dams aged between 3 to 6 months, with a history of previous pregnancies and motherhood, give birth to healthier litters. These litters can serve as a more dependable source for our animal behavioral studies. Many cases of ASD in humans are caused by non-genetic factors such as environmental influences like pesticides, air pollution, and the use of certain drugs during pregnancy. In cases of human ASD triggered by non-genetic factors, there is an increase in proBDNF, the precursor of BDNF. However, this proBDNF does not efficiently convert to BDNF. With insufficient BDNF and TrkB receptors, molecules like Akt (protein kinase B, also PKB) and Erk (Extracellular Signal-Regulated Kinase), which are crucial for neuron communication, are also less active downstream. This imbalance disrupts neuron connections, leading to ASD behaviors. In our research, the ASD-causing chemical which we used is valproic acid. It is originally an anti-seizure medication. When pregnant women took valproic acid, the chance of their child having ASD increased. Scientists used this information to inject pregnant mice with valproic acid, and as a result, all the offspring showed ASD-like behaviors. We anticipated that by isolating the brains of these offspring and measuring protein levels of BDNF, TrkB, Akt, and Erk, we would observe a similar pattern to that seen in humans with non-genetic ASD cases. We focused on studying the cortex, a region of the brain responsible for regulating social behaviors in both mice and humans. Since ASD is associated with challenges in social behaviors, we isolated the cortex from mouse brains to analyze protein levels. A chemical known as LM22A-4 with a structure resembling BDNF can bind to TrkB and activate it. We expected that the offspring of pregnant dams injected with valproic acid, which led to reduced TrkB axis activation in their brains, would show improvement in ASD behavior. This anticipation stems from the understanding that LM22A-4 activates the TrkB axis, thus compensating for its reduction, which is thought to be causing ASD-like behaviors. The offspring of mothers injected with valproic acid exhibited ASD-like behaviors, unlike the control mice. Control mice were offspring of pregnant dams injected with a solution containing only the substances used to dissolve valproic acid, typically water and salt (saline). Mice prenatally exposed to valproic acid (VPA) exhibited ASD-like behaviors, but treatment with LM22A-4 helped alleviate these behaviors, promoting more typical behavior patterns. LM22A-4, by activating TrkB receptors, helped to protect the brain from harm caused by exposure to valproic acid before birth. This could mean that valproic acid-induced changes in TrkB-related molecular mechanisms are involved in social behavior difficulties and increased repetitive behaviors seen in autism. Nevertheless, the levels of TrkB, BDNF, proBDNF, Akt, and Erk in the cortex of offspring from mothers injected with valproic acid were like those in the offspring from mothers injected with the saline solution. Therefore, the BDNF and TrkB signaling pathways remained unchanged in the cortex of our valproic acid model in this study, and they differ from those observed in human idiopathic ASD. We also speculated that a protein, called NURR1 acting upstream of BDNF and TrkB might be involved in the process. NURR1 acts as a regulatory protein that binds to the BDNF, increasing the production of copies from the BDNF. We also used a small RNA that targets a specific region in the Nurr1 and inhibits its protein production We anticipated a reduction in Nurr1 levels. As NURR1 acts as an upregulator of BDNF, lower levels of Nurr1 would result in decreased BDNF production. Activating NURR1 resulted in increased BDNF mRNA levels. However, when NURR1 was reduced, BDNF mRNA levels remained unaffected. This led us to conclude that if NURR1 levels decrease, other proteins may step in to maintain BDNF mRNA levels. Therefore, in the cortex, unlike in some other brain regions, the presence of NURR1 is not essential for regulating Bdnf. In summary, before inducing ASD-like behavior in mice using valproic acid, it is crucial to ensure the health of the mice. We used sexually mature mothers with prior pregnancy experience to provide a healthy baseline. We showed valproic acid induced ASD-like behaviors in mice offspring. We also observed that LM22A-4 treatment alleviated ASD-like behaviors of offspring. In our study, we demonstrated that the levels of BDNF, TrkB, Erk, and Akt proteins in the cortex of mice exposed to valproic acid were not affected. For this reason, our mouse model does not resemble human non-genetic ASD. Finally, NURR1's role in BDNF regulation varies by brain region. Lowering NURR1 did not affect BDNF mRNA levels, suggesting compensatory mechanisms. Our findings suggest new directions for further research to better understand the roles of TrkB and BDNF in non-genetic ASD. Overall, this study provides valuable knowledge that can contribute to advancing our understanding of idiopathic ASD-related molecular mechanisms.

This thesis presents the development of a Sensor Tower Platform (STP) comprised of an array of Passive Infra-Red (PIR) sensors along with a classification algorithm that enables the STP to distinguish between human intrusion, animal intrusion and clutter arising from wind-blown vegetative movement in an outdoor environment. The research was motivated by the aim of exploring the potential use of wireless sensor networks (WSNs) as an early-warning system to help mitigate human-wildlife conflicts occurring at the edge of a forest. While PIR sensors are in commonplace use in indoor settings, their use in an outdoor environment is hampered by the fact that they are prone to false alarms arising from wind-blown vegetation. Every PIR sensor is made up of one or more pairs of pyroelectric pixels arranged in a plane, and the orientation of interest in this thesis is one in which this plane is a vertical plane, i.e., a plane perpendicular to the ground plane. The intersection of the Field Of View (FOV) of the PIR sensor with a second vertical plane that lies within the FOV of the PIR sensor, is called the virtual pixel array (VPA). The structure of the VPA corresponding to the plane along which intruder motion takes place determines the form of the signal generated by the PIR sensor. The STP developed in this thesis employs an array of PIR sensors designed so as to result in a VPA that makes it easier to discriminate between human and animal intrusion while keeping to a small level false alarms arising from vegetative motion. The design was carried out in iterative fashion, with each successive iteration separated by a lengthy testing phase. There were a total of 5 design iterations spanning a total period of 14 months. Given the inherent challenges involved in gathering data corresponding to animal intrusion, the testing of the SP was carried out both using real-world data and through simulation. Simulation was carried out by developing a tool that employed animation software to simulate intruder and animal motion as well as some limited models of wind-blown vegetation. More specifically, the simulation tool employed 3-dimensional models of intruder and shrub motion that were developed using the popular animation software Blender. The simulated output signal of the PIR sensor was then generated by calculating the area of the 3-dimensional intruder when projected onto the VPA of the STP. An algorithm for efficiently calculating this to a good degree of approximation was implemented in Open Graphics Library (OpenGL). The simulation tool was useful both for evaluating various competing design alternatives as well as for developing an intuition for the kind of signals the SP would generate without the need for time-consuming and challenging animal-motion data collection. Real-world data corresponding to human motion was gathered on the campus of the Indian Institute of Science (IISc), while animal data was recorded at a dog-trainer facility in Kengeri as well as the Bannerghatta Biological Park, both located in the outskirts of Bengaluru. The array of PIR sensors was designed so as to result in a VPA that had good spatial resolution. The spatial resolution capabilities of the STP permitted distinguishing between human and animal motion with good accuracy based on low-complexity, signal-energy computations. Rejecting false alarms arising from vegetative movement proved to be more challenging. While the inherent spatial resolution of the STP was very helpful, an alternative approach turned out to have much higher accuracy, although it is computationally more intensive. Under this approach, the intruder signal, either human or animal, was modelled as a chirp waveform. When the intruder moves along a circular arc surrounding the STP, the resulting signal is periodic with constant frequency. However, when the intruder moves along a more likely straight-line path, the resultant signal has a strong chirp component. Clutter signals arising from vegetative motion does not exhibit this chirp behavior and an algorithm that exploited this difference turned in a classification accuracy in excess of 97%.