Browsing by Subject "Cerebellum"

Smooth pursuit eye movements are movements of the eyes that are used to foveate moving objects. Their precision and adaptation is believed to depend on a constellation of sites across the cerebellum, but only one region’s contribution is well characterized, the floccular complex. Here, I characterize the response properties of neurons in the oculomotor vermis, another major division of the oculomotor cerebellum whose role in pursuit remains unknown. I recorded Purkinje cells, the output neurons of this region, in two monkeys as they executed pursuit eye movements in response to step ramp target motion. The responses of these Purkinje cells in the oculomotor vermis were very different from responses that have been documented in the floccular complex. The simple spikes of these cells encoded movement direction in retinal, as opposed to muscle coordinates. They were less related to movement kinematics, and had smaller values of trial-by-trial correlations with pursuit speed, latency, and direction than their floccular complex counterparts. Unlike Purkinje cells in the floccular complex, simple spike firing rates in the oculomotor vermis remained unchanged over the course of pursuit adaptation, likely excluding the oculomotor vermis as a site of directional plasticity. Complex spikes of these Purkinje cells were only partially responsive to target motion, and did not fall into any clear opponent directional organization with simple spikes, as has been found in the floccular complex. In general, Purkinje cells in the oculomotor vermis were responsive to both pursuit and to saccadic eye movements, but maintained tuning for the direction of these movements along separate directions at a population level. Predictions of caudal fastigial nucleus activity, generated on the basis of our population of oculomotor vermal Purkinje cells, faithfully tracked moment-by-movement changes in pursuit kinematics. By contrast, these responses did not faithfully track moment-by-moments changes in saccade kinematics. These results suggest that the oculomotor vermis is likely to play a smaller role in influencing pursuit eye movements by comparison to the floccular complex.

Despite a largely static DNA sequence, our genomes are incredibly malleable. Comparative studies of chromatin features between different cell types, tissues, and species have revealed tremendous differences in how the genome is accessed, transcribed, and replicated. However, how the dynamics of chromatin accessibility contribute to development, environmental response, and disease status has only begun to be appreciated. In this work we identified chromatin accessibility changes by DNase-seq in three diverse processes: in granule neurons of the developing cerebellum, with intestinal epithelial cells in the absence of a normal microbiota, and with myelogenous leukemia cells in response to histone deacetylase inhibitor treatments. In all cases, we coupled these analyses with RNA-seq assays to identify concurrent transcriptional changes. By mapping the changes to these genome-wide signals we defined the contribution of local chromatin structure to the transcriptional programs underlying these processes, and improved our understanding of their relation to other chromatin changes like histone modifications. Furthermore we demonstrated use of the strongest accessibility changes to identify transcription factors critical for these processes by finding enrichment of their binding motifs. For a few of these key factors, depletion or overexpression of the protein was sufficient to regulate the expression of predicted target genes or exert limited chromatin accessibility changes, demonstrating the functional significance of these proteins in these processes. Together these studies have informed our understanding of the role chromatin accessibility changes play in development and environmental responses while also proving their utility for key regulator identification.

Classical models of cerebellar learning posit that climbing fibers operate according to a supervised learning rule to instruct changes in motor output by signaling the occurrence of movement errors. This model is grounded largely in studies of behaviors that utilize hardwired neural pathways to link sensory input to motor output. Yet, cerebellar output is also associated with non-motor behaviors, and recently with modulating reward association pathways in the VTA. Here, I test whether the supervised learning model applies to more flexible learning regimes and how the cerebellum processes reward related signals. I have used both classical and operant condition paradigms in combination with calcium imaging. In the operant conditioning paradigm I find that climbing fibers are preferentially driven by and more time-locked to correctly executed movements and other task parameters that predict reward outcome in a manner consistent with an unsigned reinforcement learning rule. In the classical conditioning paradigm I find distinct climbing fiber responses in three lateral cerebellar regions that can each signal reward prediction, but not reward prediction errors per se. These instructional signals are well suited to guide cerebellar learning based on reward expectation and enable a cerebellar contribution to reward driven behaviors.

Accumulating mental health research encourages a shift in focus towards transdiagnostic dimensional features that are shared across categorical disorders. In support of this shift, recent studies have identified a general liability factor for psychopathology – called the ‘p factor’ – that underlies shared risk for a wide range of mental disorders. Identifying the behavioral, neural, and genetic correlates of this general liability would substantiate its importance in characterizing the shared origins of mental disorders and help us begin to understand the mechanisms through which the p factor contributes to risk. In the current series of studies, I investigate the behavioral, neural, and genetic correlates of the p factor in two independent samples in order to better understand the mechanisms underlying a general liability for mental illness. I first investigate these correlates in the Duke Neurogenetics Study (DNS) comprised of 1,246 young adult volunteers aged 18-22. I then determine whether the correlates identified in the DNS replicate in a subsample of 481 45 year-old members of a birth cohort from the ongoing Dunedin Longitudinal Study (Dunedin). The Dunedin Study includes over 1,000 participants from Dunedin, New Zealand who have been followed since birth.

In Study 1 (Chapter 3), I show that the p factor, which previously has been identified and related to maladaptive behaviors in the Dunedin cohort, is identifiable in the DNS, a high-functioning young adult sample, and demonstrate that p factor scores map onto a broad range of dysfunctional behaviors. In Study 2 (Chapter 4), using high-resolution multimodal structural neuroimaging in the DNS, I demonstrate that individuals with high p factor scores show reduced structural integrity of pons white matter pathways and reduced gray matter volume in the occipital lobe and left cerebellar lobule VIIb, which is functionally connected with prefrontal regions supporting cognitive control. Consistent with the preponderance of cerebellar afferents within the pons, I observe a significant positive correlation between the white matter integrity of the pons and cerebellar gray matter volume associated with higher p factor scores. In Study 3 (Chapter 5), I replicate and extend these structural alterations in the Dunedin cohort. In Study 4 (Chapter 6), I demonstrate that liabilities for internalizing, externalizing, and thought disorders overlap with some structural neural correlates of the p factor, but also have unique correlates with brain structure in both samples. Finally, in Study 5 (Chapter 7), I show that polygenic risk for schizophrenia is associated with higher p factor scores, and that this association is partly mediated by cortico-cerebellar circuitry in the DNS, but not the Dunedin, sample.

Overall, these findings provide initial evidence that structural alterations in occipital and cortico-cerebellar circuitry supporting core functions related to the basic integration, coordination, and monitoring of information may contribute to a general liability for mental disorders and are robust to differences in sample characteristics. Links between genes, brain, and behavior in the DNS suggest that polygenic risk for schizophrenia may confer increased behavioral risk for general psychopathology through its influence on the capacity to communicate and process information between the cerebrum and the cerebellum through the pons. Failure to replicate these latter associations in the Dunedin cohort suggests important future research directions such as examining more specific genetic influences as well as their interactions with early life experiences on brain structure and general risk for psychopathology.

Accumulating mental health research encourages a shift in focus towards transdiagnostic dimensional features that are shared across categorical disorders. In support of this shift, recent studies have identified a general liability factor for psychopathology – called the ‘p factor’ – that underlies shared risk for a wide range of mental disorders. Identifying the behavioral, neural, and genetic correlates of this general liability would substantiate its importance in characterizing the shared origins of mental disorders and help us begin to understand the mechanisms through which the p factor contributes to risk. In the current series of studies, I investigate the behavioral, neural, and genetic correlates of the p factor in two independent samples in order to better understand the mechanisms underlying a general liability for mental illness. I first investigate these correlates in the Duke Neurogenetics Study (DNS) comprised of 1,246 young adult volunteers aged 18-22. I then determine whether the correlates identified in the DNS replicate in a subsample of 481 45 year-old members of a birth cohort from the ongoing Dunedin Longitudinal Study (Dunedin). The Dunedin Study includes over 1,000 participants from Dunedin, New Zealand who have been followed since birth.

In Study 1 (Chapter 3), I show that the p factor, which previously has been identified and related to maladaptive behaviors in the Dunedin cohort, is identifiable in the DNS, a high-functioning young adult sample, and demonstrate that p factor scores map onto a broad range of dysfunctional behaviors. In Study 2 (Chapter 4), using high-resolution multimodal structural neuroimaging in the DNS, I demonstrate that individuals with high p factor scores show reduced structural integrity of pons white matter pathways and reduced gray matter volume in the occipital lobe and left cerebellar lobule VIIb, which is functionally connected with prefrontal regions supporting cognitive control. Consistent with the preponderance of cerebellar afferents within the pons, I observe a significant positive correlation between the white matter integrity of the pons and cerebellar gray matter volume associated with higher p factor scores. In Study 3 (Chapter 5), I replicate and extend these structural alterations in the Dunedin cohort. In Study 4 (Chapter 6), I demonstrate that liabilities for internalizing, externalizing, and thought disorders overlap with some structural neural correlates of the p factor, but also have unique correlates with brain structure in both samples. Finally, in Study 5 (Chapter 7), I show that polygenic risk for schizophrenia is associated with higher p factor scores, and that this association is partly mediated by cortico-cerebellar circuitry in the DNS, but not the Dunedin, sample.

Overall, these findings provide initial evidence that structural alterations in occipital and cortico-cerebellar circuitry supporting core functions related to the basic integration, coordination, and monitoring of information may contribute to a general liability for mental disorders and are robust to differences in sample characteristics. Links between genes, brain, and behavior in the DNS suggest that polygenic risk for schizophrenia may confer increased behavioral risk for general psychopathology through its influence on the capacity to communicate and process information between the cerebrum and the cerebellum through the pons. Failure to replicate these latter associations in the Dunedin cohort suggests important future research directions such as examining more specific genetic influences as well as their interactions with early life experiences on brain structure and general risk for psychopathology.

During development, the precise regulation of the processes of proliferation, migration, and differentiation is required to establish proper organ structure and function and to prevent the deregulation that can lead to disease, such as cancer. Improved understanding of the signals that regulate these processes is therefore necessary to both gain insight into the mechanisms by which organ development proceeds and to identify strategies for treating the consequences of deregulation of these processes. In the cerebellum, some of the factors that regulate these processes have been identified but remain incompletely understood. Our studies have focused on the signals that regulate the migration of cerebellar granule neuron progenitors (GNPs) and the contribution of the SDF-1/CXCR4 signaling axis to postnatal cerebellar development. Using conditional knockout mice to delete CXCR4 specifically in GNPs, we show that loss of CXCR4 results in premature migration of a subset of GNPs throughout postnatal development that are capable of proliferation and survival outside of their normal mitogenic niche. Loss of CXCR4 also causes a reduction in the activity of the Sonic hedgehog (SHH) pathway (the primary mitogen for GNPs) but does not affect GNP proliferation, differentiation, or capacity for tumor formation. Our data suggest that while other factors likely contribute, SDF-1/CXCR4 signaling is necessary for proper migration of GNPs throughout cerebellar development.

In addition to understanding the signals that regulate normal development, the identification of vulnerabilities of established tumors is also necessary to improve cancer treatment. One strategy to improve treatment involves targeting the cells that are critical for maintaining tumor growth, known as tumor-propagating cells (TPCs). In the context of the cerebellar tumor medulloblastoma (MB), we have previously identified a population of TPCs in tumors from patched mutant mice that express the cell surface carbohydrate antigen CD15/SSEA-1. Here, we employed multiple approaches in an effort to target these cells, including a biochemical approach to identify molecules that carry the CD15 carbohydrate epitope as well as an immunotoxin approach to specifically target CD15-expressing cells. Unfortunately, these strategies were ultimately unsuccessful, but an alternative approach that recognized a vulnerability of CD15+ cells was identified. We show that CD15+ cells express elevated levels of genes associated with the G2/M phases of the cell cycle, progress more rapidly through the cell cycle than CD15- cells, and contain an increased proportion of cells in G2/M. Exposure of tumor cells to inhibitors of Aurora and Polo-like kinases, key regulators of G2/M, induces cell cycle arrest, apoptosis and enhanced sensitivity to conventional chemotherapy, and treatment of tumor-bearing mice with these agents significantly inhibits tumor progression. Importantly, cells from human patient-derived MB xenografts are also sensitive to Aurora and Polo-like kinase inhibitors. Our findings suggest that targeting G2/M regulators may represent a novel approach for the treatment of human MB.

Examining the coordination of excitatory and inhibitory (E/I) activity within cortical circuits is a fundamental approach to understanding normal and aberrant circuit function. Alteration to this E/I coordination is one of the leading pathophysiological models for several neurological disorders, including epilepsy, schizophrenia, ADHD, and autism spectrum disorders (ASD) (Nelson et al. 2015, Mullins et al. 2016). The overarching goal of this research is to understand the mechanism that regulates excitatory and inhibitory coordination within the cerebellum. Using a range of techniques to monitor and manipulate cortical circuits; I examined how the neuromodulator, acetylcholine, alters E/I activity at the initial input stage of the cerebellar cortex, the granular layer. Additionally, in a collaboration with Dr. Hatten's group at Rockefeller, we investigated the post-migratory role of Astrotactin 2 (ASTN2), a risk gene for autism; and found a role in regulating the surface level expression of synaptic proteins. By investigating how basic circuit function is modified in a transient manner, i.e. neuromodulation, we can reveal mechanisms that facilitate context-dependent learning; moreover, by examining permanent modifications, e.g. ASTN2, to circuit function, we can begin to understand neural mechanisms that underlie both normal synaptic function and the pathophysiology of ASDs.

These studies revealed the following: 1) acetylcholine actively modifies two out of three main nodes - excitatory mossy fiber terminals and inhibitory Golgi cells - within the granule cell layer. This modulation resulted in a bidirectional change in the excitability of granule cells, suggesting that cholinergic circuits within the granule cell layer are well situated to alter information processing in a context-specific manner. 2) ASTN2 binds and regulates the surface expression of multiple synaptic proteins via endocytosis. A truncated form of ASTN2 (ASTN2-JDUP), resulting from copy number variation in its FNIII and MAC/Perforin domains, occurs in patients with neurodevelopmental disorders. Investigations into this JDUP variant revealed changes in the binding affinity to several different binding partners, including neuroligins 1-4. Additionally, conditional overexpression of JDUP or ASTN2 in Purkinje cells, revealed differential changes in postsynaptic glutamatergic and GABAergic activity. Overall, these ASTN2 results lay the foundation for future studies using an ASTN2 loss-of-function mouse model.