The development of cholinergic neurons in mouse frontal cortex was studied both in vivo and in vitro by immunocytochemistry with an antibody to choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine synthesis. While cortical cholinergic neurons have previously been characterized in rat cortex, up until very recently, intrinsic cortical cholinergic neurons were considered to be absent in mouse, and little is known about their development or phenotypic characteristics. The present study found no ChAT-positive neurons in mouse frontal cortex on postnatal day 0 (P0, the day of birth). On P7 there were few, faintly stained, ChAT-positive neurons. The numerical density of ChAT-positive neurons increased substantially with age, from none on P0, to 9.2 + 1.4 on P7, to 14.8 + 0.9 on P16, and 41.6 + 3.9 in adulthood. Considering that the numerical density of total neurons decreases during this postnatal period, the data represent a marked developmental increase in the percentage of cholinergic neurons. The development of cholinergic neurons showed very similar timelines in rat and mouse frontal cortex. Cultures prepared from mouse frontal cortex on embryonic day 16 were maintained for 25, 76, or 100 days in vitro (div). The percentage of ChAT-positive neurons was considerably higher than in vivo, ranging from a mean 28% to 31% across the three age (div) groups. With increasing age of the cultures, the numerical density of total neurons and ChAT-positive neurons decreased while the percentage of ChAT-positive neurons did not change significantly. These observations suggest some temporal stability in the cultures. Using dual immunofluorescence, ChAT-positive neurons were tested for colocalization with GAD or TH. The majority of ChAT-positive neurons colocalized with GAD, both in vitro and in vivo. However, ChAT did not colocalize with TH, either in vitro or in vivo. Our comparison of intact frontal cortex and cultures suggest that while the percentage of cholinergic neurons was greater in the cultures, the cholinergic neurons developed phenotypic similarities in vitro and in vivo.

Memory is critical to our everyday lives, allowing us to attach meaning to our experiences of the world. However, a number of neurocognitive disorders can result in the loss of this fundamental function. The development of effective treatments for loss of episodic memory depends on a detailed understanding of the neural signals that support memory and a thorough characterization of how brain stimulation may be targeted to modulate memory-related patterns of brain activity. In this dissertation, I approach these questions with a series of three studies to examine the effects of direct electrical brain stimulation, the role of large-scale patterns of brain activity in memory, and how stimulation can be used to modulate these signals. In my first study, I characterize changes in neuronal activity across the brain that resulted from delivering stimulation at a range of frequencies, amplitudes, and locations. To do this, I developed an analysis framework and applied it to a large-scale dataset of direct human brain recordings from electrodes implanted in neurosurgical epilepsy patients while intracranial stimulation was delivered. With these analyses, I found that stimulation most often had an inhibitory effect; however, high-frequency stimulation delivered near white-matter tracts was most likely to excite neuronal activity. In my second study, I investigated the functional role of brain oscillations that moved across the cortex during memory tasks. I found that traveling waves of low-frequency oscillations that moved anteriorly across the cortex most often supported successful memory encoding. Additionally, the timing, or phase, of brain oscillations propagating across specific areas of the cortex predicted efficient memory retrieval. In my last study, having determined that the direction of traveling waves is important for memory processes, I then investigated how different types of stimulation changed the direction of traveling waves of low-frequency oscillations. By analyzing intracranial recordings during a stimulation mapping procedure, I found that stimulation at high frequencies oriented in line with the direction of wave propagation was most effective in changing the propagation direction of traveling waves. Additionally, I tested how changes traveling wave direction from stimulation affected patients’ memory performance during an episodic memory task. For patients where stimulation changed the propagation direction of their waves from anterior to posterior directions, stimulation also impaired their memory, and when stimulation had the opposite effect on direction, it enhanced their memory. This provides the first preliminary causal evidence that stimulation can be targeted to modulate specific features of large-scale patterns of brain oscillations— the direction of traveling waves— and, in turn, affect memory performance. Broadly, this body of work shows that direct electrical stimulation of the brain applied with specific parameters holds the potential to modulate neural activity related to memory. This work expands our current understanding of the functional role of brain oscillations by showing that specific features of traveling waves across the cortex are key signals linked to human behavior. These findings provide both a basic understanding of how neural oscillations support human behavior as well as a foundation for designing stimulation protocols to precisely target desired changes in neural activity with the potential to improve diagnostic and therapeutic applications.

In this thesis I present novel mechanisms for certain computational capabilities of the cerebral cortex, building on the established notion of attractor memory. A sparse binary coding network for generating efficient representation of sensory input is presented. It is demonstrated that this network model well reproduces receptive field shapes seen in primary visual cortex and that its representations are efficient with respect to storage in associative memory. I show how an autoassociative memory, augmented with dynamical synapses, can function as a general sequence learning network. I demonstrate how an abstract attractor memory system may be realized on the microcircuit level -- and how it may be analyzed using similar tools as used experimentally. I demonstrate some predictions from the hypothesis that the macroscopic connectivity of the cortex is optimized for attractor memory function. I also discuss methodological aspects of modelling in computational neuroscience. / QC 20101220

Through mutations in the genes TSC1 and TSC2, the genetic disorder Tuberous Sclerosis Complex (TSC) causes begin tumors to develop in different organs across the body. Of the many ways that this disorder can manifest, the brain is one of the most commonly affected organs in TSC. Mutations in TSC1 or TSC2 result in mTORC1 hyperactivation and can impact how the brain forms early in development. Most patients with TSC exhibit seizures and over half display some level of intellectual disability, highlighting the impact that mTORC1 hyperactivation can have on brain function and cognition. However, despite our understanding of the genetic cause of TSC, the mechanisms downstream of TSC1/TSC2 and mTORC1 that mediate TSC neuropathology are not well understood. Therefore, additional study of the cellular and molecular underlying the aberrant neurodevelopment found in TSC and other mTOR-overactivation disorders (collectively known as mTORopathies) is necessary for further understanding of these disorders. Of the pathways that have been identified to interact with mTORC1, there has been great interest in understanding the relationship between mTORC1 and Hippo-YAP/TAZ signaling. The Hippo pathway is an evolutionarily considered pathway that is crucial for regulating organ size through its control of the transcriptional co-activators YAP/TAZ. As exhibited through study of the murine brain, hyperactivation of YAP/TAZ causes changes in how the cortex develops, with several features overlapping with mTORC1 hyperactivation (including aberrant neuronal migration, changes in neuron structure, and increased progenitor proliferation). While the relationship between mTORC1 and YAP/TAZ has been explored in other systems, its connection in the brain has yet to be explored. In Chapter 1 of this dissertation, I first review how TSC affects cortical development as a whole by addressing what is known about the specific cell types and signaling pathways that are affected this disorder. Of the signaling pathways described, the Hippo- YAP/TAZ pathway is discussed in particular detail, addressing its role not only in the context of TSC and in terms of its interaction with mTORC1 signaling, but also in terms of its general role in cortical development. In discussing these studies, I describe the phenotypes seen in different mouse models and in the human brain, allowing for the identification of pathological features that are common between species and between different Cre lines. Following this initial review, I present our experimental aims, hypotheses, and experimental overview for this project in Chapter 2. In Chapter 3, I describe our investigation into the role of YAP/TAZ in the abnormal neurodevelopment that occurs in TSC. Through our analysis of human cortical tuber samples, I demonstrate that YAP/TAZ are elevated at the protein level and that two of their established target genes, CYR61 and CCN2, are elevated at the mRNA and protein levels. Having demonstrated that YAP/TAZ levels and activity are elevated in cortical tuber samples, I next went on to establish whether YAP/TAZ are similarly changed in our TSC animal model. Examination of Emx1-Cre driven Tsc2 cKO mice showed that the level of Yap/Taz were significantly elevated at E16.5. Having established that both YAP/TAZ levels are elevated in our animal model, I next sought to determine whether concurrent genetic manipulation of Yap/Taz in our Tsc2 cKO animals would reduce the severity of neuropathology seen in these mice. Triple conditional knockout (tcKO) of Yap/Taz/Tsc2 was sufficient to mitigate several features seen with mTORC1 hyperactivation in the brain, including the cortical thickness increases, abnormal neuronal migration in the cortex, hippocampal lamination defects, and hypomyelination found in their single Tsc2 cKO counterparts. Overall, these findings provide additional evidence that mTORC1 hyperactivation positively regulates YAP/TAZ. For the first time, this study describes elevation of YAP/TAZ in the brains of individuals with TSC and in the brains of a TSC mouse model. Furthermore, I provide evidence that reduction of Yap/Taz may have a beneficial effect on neuropathology in TSC, highlighting an area for future research in the development of novel therapeutics for this disorder. / Biomedical Sciences

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Using functional magnetic resonance imaging in humans, Brodmann's area 5 (BA5) is observed to be activated during the suppression of motor output in the context of a NO-GO task. In monkeys, BA5 is associated with somatosensation and specifically linked with motor preparation. The goal of this thesis is to investigate BA5 influences on corticospinal excitability prior to the onset of movement, in the context of a GO/NO-GO paradigm. To achieve this goal, paired-pulse TMS is used to probe the functional connectivity between BA5 and ipsilateral primary motor cortex (M1) for a muscle specific to the hand. Three experiments are performed that investigate the differences in corticospinal output to the hand in a GO task versus a NO-GO task and the stimulation parameters that reveal such differences. Results indicate that BA5 is able to condition M1 prior to movement in a task-specific manner. Further, motor evoked potentials (MEPs) are suppressed in the context of a NO-GO task relative to a GO task, and task-specific differences rely on the intensity and direction of induced current in the cortex. In conclusion, data from this thesis contribute to our understanding of the role of BA5 in motor control. / Thesis / Master of Science in Kinesiology

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