Genetic dissection of loci important in the control of neurogenesis has improved our understanding of both the evolutionarily conserved and divergent processes in neurodevelopment. These loci include not only protein coding genes [1, 2], but also noncoding RNAs [3-5]. One important family of non-coding RNAs is miRNAs, which control gene expression fundamental in developmental regulation and mature cell maintenance [3, 5-9]. Here, we will first focus our efforts by surveying miRNA regulation in the developing brain. We hypothesize a strong regulatory role of miRNAs during proliferation, cell death, migration and differentiation in the developing mammalian forebrain that has yet to be adequately described in the literature. Second, we will assess miRNA's role in the evolutionary divergence of brain-related gene expression. We hypothesize that a human specific single nucleotide change(s) in the miRNA recognition element of transcription factors 3' untranslated regions contributes to species-specific differences in transcription factor expression and ultimately alters regulatory function.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a mutation involving an expansion of the CAG trinucleotide repeats in the gene encoding for huntingtin (HTT) protein. The discovery of the disease-causing faulty gene (mutant huntingtin; mHTT) has enabled valid presymptomatic gene assessment for HD with results being categorized as ‘gene-expanded (GE; CAG repeats ≥ 36)' or ‘gene non-expanded (GNE)'. Individuals tested to be gene-expanded are destined to develop HD symptoms and will receive clinical diagnosis at an average age of 40 years when abnormal motor symptoms manifest. Those who are GNE will not develop HD. The availability of genetic testing has also provided a valuable research opportunity to study the pathoetiology of HD in PreHD subjects (those tested to be ‘gene-expanded' but are in the prediagnostic stage of HD). The genetic mutation results in widespread neuronal degeneration preferentially within the striatum. The clinical manifestations of HD include a triad of motor, cognitive and psychiatric symptoms. Challenging the classical view of HD as a neurodegenerative disease, recent studies have brought about a conceptual shift to include abnormal neurodevelopmental aspects in the etiology of HD1, based on the notion that lifelong HTT gene mutation may compromise HTT's crucial role in normal brain development. The fronto-striatal circuitry has been a main interest in HD research for its profound pathological association with symptom manifestation and marked neuroanatomical change. However, no study to date has investigated the neuropathological alteration of the fronto-striatal circuitry during childhood in mHTT carriers. In line with the proposed new perspective, the overall hypothesis of the current proposal is that the deteriorating effect of mHTT on the fronto-striatal circuits stems from abnormal development of these circuits. Therefore, the main goal of the current study was to enhance our understanding of how mHTT alters the evolving capacity of the fronto-striatal circuitry from a developmental perspective. To this end, the study examined the fronto-striatal circuit structure (using magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI)) and function (using resting state functional MRI and cognitive/behavior tests) in children (6-18 years of age) at risk for HD. Healthy control children with no family history of HD were also evaluated. For research purposes only, children at risk for HD were genotyped and designated as GE or GNE based the result of their genetic testing. While the assessment of the fronto-striatal circuit development focused on anatomical delineations, the tests of the fronto-striatal circuit functionality were carried out separately for the three distinct cognitive, motor and affective loops within the fronto-striatal circuitry. The overall aim of the project was to evaluate the effect of mHTT on subcortical brain structures, white matter connection development and functional integrity of the cognitive, motor and affective loops within the fronto-striatal circuitry in Gene-Expanded (GE) children compared to those of Healthy Control (HC) children and again to those who are Gene Non-Expanded (GNE). Test of the hypothesized mHTT-associated developmental alteration of the fronto-striatal circuitry was addressed in two specific aims: Specific Aim #1: To observe subcortical gray matter volumes and white matter integrity of the fronto-striatal circuitry in children at risk for HD Decline in corpus striatal volume as well as aberrant fronto-striatal circuit connectivity has been reported in manifest HD patients and also in preHD adults.2-4 However, there is no experimental evidence of the onset or pattern of the pathophysiological change in the fronto-striatal circuitry. Therefore, volumes of subcortical structures and white matter integrity were measured in order to assess the development of the fronto-striatal circuitry in children at risk for HD. Specific Aim #2: To assess the functionalities of specific fronto-striatal circuit loops in children at risk for HD Functional abnormalities of the fronto-striatal circuitry have been observed in HD patients as well as in preHD adults.5 In order to closely examine the mHTT effect on fronto-striatal circuit function, resting state functional connectivity as well as performances on cognitive and behavioral tasks tapping into the functionality of the three fronto-striatal loops were evaluated in children at risk for HD. It was hypothesized that GE children would have diminished fronto-striatal circuit function. The GE children were predicted to show statistically significant deficits in 1) resting state functional connectivity between specific frontal lobe areas and striatal sub-regions, 2) cognitive control, 3) motoric control and 4) behavior control when compared to healthy controls and children without the HTT CAG expansion. The current study reports the baseline profile of the fronto-striatal structure and function in children at risk for HD. We have found that children who are on average 30 years ahead of HD diagnosis to have developmental alterations in the brain structure and function directly linked to the effect of mHTT. Brain morphology analysis revealed specific subcortical gray and white matter changes. The changes include disproportionately smaller caudate and putamen volumes and increased radial diffusivity localized to the external capsule which were more evident in males with HTT gene expansion. Fronto-striatal circuit functional assessment revealed a drop in motor functionality (both at rest and active performance) and externalizing behavior problems indicative of compromised inhibition than aggression. Children from HD families but do not have the genetic mutation also showed development aberrations in both brain structure and function when compared to healthy controls. Importantly, the altered structural morphology and functional profile seen in the GNE group differed from that of the GE children, emphasizing the impact of mHTT. The findings from those who share similar household environment but differ in genetic expansion status are important in highlighting the potential interaction of gene-environment effect on the manifestation of mHTT related changes seen in the children with the genetic expansion for HD. Investigation of subtle but persistent effects of mHTT on normal neural developmental processes may further our understanding of the pathogenesis of HD. Continuous longitudinal comprehensive assessments of the mHTT associated neurophenotype would aid in prognostic scenario estimation and thereby lead to effective clinical decision making to maximize the benefit of early intervention.

Breathing is an essential homeostatic function and its disruption leads to disability, brain damage, and death. Serotonin (5-hydroxytryptamine; 5-HT) neurons in the brainstem play an important role in control of breathing. Medullary 5-HT neurons are stimulated by increased CO₂ and subsequently stimulate respiratory nuclei to increase ventilation and maintain normal blood gas levels. Anesthetic-induced breathing dysfunction is a serious concern in healthcare settings. In research settings, experiments are often performed under anesthesia, and therefore it is important to understand how these drugs affect animal physiology. Unfortunately, little is known about how anesthetics modulate 5-HT neurons, breathing, and CO₂ chemoreception in mice, as many of the previous studies have been performed in different species. Characterizing how anesthetics commonly used in both research and clinical settings affect 5-HT neurons, breathing and CO₂ chemoreception is valuable to the broader field of neuroscience since these drugs are so ubiquitously used in research. Breathing dysfunction and defects in the serotonergic system have been implicated in disorders, such as sudden unexpected death in epilepsy (SUDEP) and sudden infant death syndrome (SIDS), which means better characterizing the role of 5-HT neurons in breathing has translational impact as well. Here I examine whether halogenated inhalational anesthetics, which potentiate TWIK-related acid-sensitive K⁺ (TASK) currents and GABAA receptors, could mask an effect of CO₂ on 5-HT neurons. During in vivo plethysmography in mice, a therapeutic level of isoflurane (1%) markedly reduced the hypercapnic ventilatory response (HCVR) in all mouse strains tested. In dissociated cell cultures, isoflurane (1%) hyperpolarized 5-HT neurons and inhibited spontaneous firing. A subsequent decrease in pH from 7.4 to 7.2 depolarized 5-HT neurons, but that was insufficient to reach threshold for firing. Depolarizing current restored baseline firing and the firing frequency response to acidosis, indicating that isoflurane did not block the underlying mechanisms mediating chemosensitivity. These results demonstrate that isoflurane masks 5-HT neuron chemosensitivity in vitro, and markedly decreases the HCVR in vivo. Next, I demonstrate that ketamine-xylazine or urethane anesthesia also significantly reduced the HCVR in mice at both therapeutic and sub-therapeutic doses. However, mice treated with a sub-therapeutic dose of anesthesia decreased their O₂ consumption in parallel, and thus matched their ventilation to metabolic demands. Mice that were anesthetized with the therapeutic dose did not sufficiently match their breathing and metabolic demands, and thus anesthesia induced hypoventilation. Recordings from 5-HT neurons in culture indicated that neither ketamine nor urethane affected 5-HT neuron chemosensitivity. These data demonstrate that anesthetics with different molecular targets similarly reduce the HCVR in mice, but not all of their effects are mediated via 5-HT neurons. Moreover, both ketamine-xylazine and urethane anesthesia altered baseline breathing in different ways, suggesting they targeted different parts of the respiratory network. Finally I show that isoflurane anesthesia in neonatal mice caused depression of resting ventilation, which was different from isoflurane-anesthetized adults. This effect was more pronounced in wildtype mice compared to littermates with genetic deletion of 5-HT neurons. Isoflurane-induced breathing depression decreased and mice fully recovered following washout of isoflurane at P8. I observed that genetic deletion of 5-HT neurons in mice with a congenic C57Bl/6 background led to a more severe phenotype than previously described in mixed genetic background strains. These mice had decreased survival, severe growth retardation, and reduced baseline ventilation. These results indicate that 5-HT neurons have a different role during the neonatal period and that some mouse strains are more sensitive to genetic deletion of 5-HT neurons; thus, background genetics play an important role in phenotype presentation. In summary, different classes of anesthetics each strongly depress chemoreception. Isoflurane seems to affect breathing, in part, by hyperpolarizing 5-HT neurons and masking their chemosensitivity, whereas ketamine and urethane have less effect on 5-HT neurons. However, both ketamine-xylazine and urethane anesthesia alter baseline breathing. Isoflurane anesthesia decreases baseline ventilation in neonates, but this effect is absent in adults, which suggests that the effects of isoflurane on breathing changes as mice age. These data are important for the field of respiratory physiology because they highlight the sensitivity of breathing to the effects of anesthetics. These results are valuable to the broader field of neuroscience, because anesthetics are widely used during in vivo research. Additionally, some transgenic mouse strains are more sensitive to 5-HT neuron deletion depending on their genetic background. In the future it will be critical to characterize the molecular mechanisms that underlie these phenomena.

Spinocerebellar ataxia type 1 (SCA1) is an adult onset, autosomal dominant neurodegenerative disease caused by a CAG repeat expansion in ataxin-1, which encodes the ataxin-1 protein. SCA1 is one of nine polyQ-expansion gain-of-function diseases which includes Huntington's disease, spinal-bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy and other ataxias. Clinical symptoms of SCA1 include ataxia, dysarthria, ophthalmoparesis, muscle wasting, and extrapyramidal and bulbar dysfunction. Cerebellar Purkinje cells (PCs), neurons in the inferior olive and nuclei of the brainstem are affected. No disease-modifying therapy exists for SCA1. The goals of my thesis were to assess the safety and efficacy of AAV-delivered artificial miRNAs targeting ataxin-1 to alleviate neuropathological and behavioral phenotypes in the knock-in and transgenic SCA1 mouse models. In the knock-in SCA1 mouse model AAVs expressing an artificial miRNA (miSCA1) targeting sequences conserved in mouse and human ataxin-1 were injected directly to the deep cerebellar nuclei. This achieved long term silencing of ataxin-1 mRNA and significantly improved rotarod performance, gait deficiencies, and neuropathology of the cerebellum. In the transgenic SCA1 mouse model the same method of delivery was executed with an artificial microRNA (miR) (miS1) designed to optimize potency, efficacy and safety to suppress Atxn1 expression. Additionally the therapeutic potential of continuous overexpression of ataxin-1-like was examined. Delivery of either ataxin-1-like or miS1 viral vectors to SCA1 mouse cerebellum resulted in widespread cerebellar Purkinje cell transduction. There was significant improvement to rotarod performance, gait deficiencies, coordination and balance, as well as the neuropathology of cerebellar Purkinje cells. In summary, these data indicate the utility of these approaches as possible therapies for SCA1 patients.

Attention Deficit Hyperactivity Disorder, a disorder marked by a pattern of inattention and hyperactivity, is commonly treated with the drug methylphenidate (MPH), which inhibits reuptake of the neurotransmitters norepinephrine and dopamine, thereby increasing the levels of these catecholamines in the synaptic cleft. In addition, MPH is abused by students studying for exams to increase focus and wakefulness. Despite the extensive use of MPH, little is known its long-term effects on the brain. In this study, we examined the impact of 4 weeks of MPH treatment on neurogenesis or the “birth” of new brain cells in the hippocampus of male adolescent mice. Neurogenesis was measured using 5’-ethinyldeoxyuridine (EdU), a thymidine analog that gets incorporated into DNA before cell division, and total neuron numbers were estimated using the neuronal marker, NeuN. Interestingly, low (1 mg/kg) and high (10 mg/kg) doses of MPH delivered twice daily, increased the rate of neurogenesis after 4 weeks. We also examined the survival of the new cells 4 weeks after EdU injection, both with and without continued MPH treatment. Cell counts were performed, and ratios of EdU+/NeuN+ cells were compared. Although both 1 mg/kg and 10 mg/kg MPH increased the ratio of EdU+/NeuN+ cells, the EdU+/NeuN+ ratios were no different from control if MPH was not continued. If low dose of MPH was continued for an extra 4 weeks, survival of newly generated cells was enhanced; this was not the case for the high dose of MPH. To investigate the mechanism for MPH-induced changes in hippocampal neurogenesis, we examined the levels of proteins linked to cell growth and survival in the hippocampus, including brain derived neurotrophic factor (BDNF), glial cell line derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF), tropomyosin receptor kinase B (TrkB, the receptor for BDNF) and beta-catenin. Levels of BDNF or GDNF were examined using enzyme-linked immunosorbent assays (ELISAs), and VEGF, TrkB, and beta-catenin expression was investigated using simple western. Interestingly, 1 mg/kg MPH appears to increase VEGF, TrkB, and beta catenin after 4 weeks. In animals treated with 10 mg/kg MPH, despite the increases in neurogenesis after 4 weeks of treatment, beta catenin levels decreased compared to control at 4 weeks, and VEGF, TrkB and beta catenin levels were decreased at 8 weeks. Thus, long-term exposure to MPH increases neurogenesis rate in the hippocampus, and the effect of low doses of MPH may be related to the increased expression of VEGF, TrkB and beta catenin.

Language has been the subject of academic fascination for centuries, and the ability to communicate abstract notions through speech and writing allows humans to interact in ways that would not otherwise be possible. While the mechanisms of language processing have been studied extensively with behavioral and noninvasive neuroimaging methods, much about how the brain encodes language remains unknown. In this dissertation, I describe experiments using intracranial neurophysiology in humans to interrogate the mechanisms of language perception at high spatiotemporal resolution. First, I explore the neural mechanisms of visual word recognition in a large human intracranial dataset. By analyzing population sensitivity to a hierarchy of word features, I create a high-resolution map of stimulus encoding during single-word reading that reveals the early influence of lexical features in lingual and fusiform gyri followed by a cascade of lexical, orthographic, and semantic information in temporal and frontal lobes. Along with clustering analyses that show stimulus encoding in anatomically distributed populations, these results demonstrate that feed-forward, feed-back, and distributed processing mechanisms underlie visual word recognition. Second, I describe the development of an artificial language task designed to characterize the neural mechanisms of auditory word segmentation. The task is designed in three phases to probe how the brain tracks distributional regularity and the neural mechanisms of word segmentation with and without lexical access. Taken together, this work expands our understanding of the neural mechanisms of language processing using human intracranial neurophysiology.

Within five weeks, the nudibranch, Berghia stephanieae (Gastropoda, Mollusca) can regenerate a severed rhinophore, the main olfactory appendage, such that it is indistinguishable from a non-lesioned rhinophore. The rhinophore is a 2 mm long stalk with lateral sides covered in ridges and the distal third covered in fluorescent pigmentation. Its internal morphology is dominated by longitudinal musculature, overlaid by a complex neuronal plexus, which contains neurons that express various neuroactive substances including serotonin and small cardioactive peptide (SCP). Two large nerves originate in the rhinophore ganglion at the base of the rhinophore and run the length of the rhinophore. To quantify the regeneration of these external and internal characteristics, the rhinophore was entirely severed, with animals then sacrificed and dissected at one-day, three-days, five-days, seven-days, 21-days, and 35-days post-lesion. Immunohistochemical and neuronal tracing techniques were used to study the neuroanatomy of the regenerated structure. At each time point, brains, consisting of the cerebral, pleural, and pedal ganglia with attached rhinophores were quadruple stained with 4′,6-diamidino-2-phenylindole (DAPI) and phalloidin to label nuclei and actin respectively, and immunolabeled for SCP and serotonin. The length of regenerated rhinophores was measured and compared with the unlesioned side. There was a period of disorganization in the first five days, followed by rapid increase in size and a gradual slowing of growth from 21 days to 35 days. Ridge reappearance followed a similar trajectory, regrowing from the base of the rhinophore, rapidly after the first five days with gradual slowing and not matching the extent seen on unlesioned appendages. Fluorescent pigmentation on the distal part of the rhinophore was the slowest feature to return, first visible at 21-days post-lesion. These external features were still not equivalent to those seen in unlesioned rhinophores at 35 days. Internally, phalloidin labeling showed that the muscles were disorganized during the first week, but then regrew, matching the return of the external anatomy. Muscle fiber thickness did not vary between regenerated and unlesioned rhinophores. The SCP and serotonin immunoreactivity exhibited different patterns of regrowth. Serotonin fibers were present immediately after lesion at the tip of the rhinophore and increased in density over the rest of the rhinophore with increasing time post-lesion. The SCP plexus, in contrast, was not present until a week after lesion, and at later timepoints there was not a consistent pattern. Lastly, the projections into and from the brain were traced by application of neurobiotin to the cut ends of the rhinophore nerves. There was no obvious difference between regenerated and unlesioned sides five-weeks after the removal of one rhinophore; all major neural tracts were present, including those that traveled from the regenerated side contralaterally through the brain and up into the contralateral rhinophore ganglion. The neuroanatomy of Berghia’s brain and rhinophores thus suggests that after complete severance of one rhinophore, the animal is able to regrow the cells and fiber tracts in five weeks.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / While memory function in primates depends on the integrity of the medial temporal lobe, the contribution of the hippocampal formation (HF) independent of the overlying ventromedial temporal cortices, particularly the entorhinal (ENT) and parahippocampal (PHG) cortices, remains unclear. To address this issue we have prepared groups of rhesus monkeys with ibotenic acid lesions of the HF or aspiration lesions of the ENT or PHG cortices. We then administered behavioral tasks to assess the effects of these lesions relative to normal controls. To test recognition memory, the Delayed Non-Matching to Sample (DNMS) task and the Delayed Recognition Span Task (DRST) were administered. On DNMS, all groups were impaired on both acquisition and 2 and 10 minute delays. The DRST, administered in Spatial, Color and Object conditions, yielded slightly different results. On the Spatial condition, all groups were impaired on both unique and repeated trials of the task. On the Color condition, all groups were impaired on unique trials while only the HF group was impaired on repeated trials. On the Object condition, ENT and PHG groups were only impaired on unique trials, while the HF group was unimpaired. To assess associative memory, two choice reversals were administered in Spatial (SR) and Object (OR) modalities. On the SR task, The HF group was impaired on acquisition and the first of three reversal phases. The ENT group was impaired on all three reversals, and the PHG group was impaired on only the last. On the OR task, HF animals were impaired on all reversals, while ENT animals were impaired on the initial reversal and PHG animals on the last two. These results indicate that damage to the HF alone causes impairments in recognition, spatial processing and object reversal learning. They also indicate that ENT and PHG regions make unique contributions to memory processes as seen in additional impairments on DRST and the inability to perform spatial reversals. Thus impairments previously attributed to hippocampal damage in studies where the ENT and PHG cortices were removed in conjunction with the HF need to be reevaluated in view of additional contributions provided by these cortical regions. / 2031-01-01

Schizophrenia is a debilitating psychiatric disorder that affects approximately 1% of the global population. Schizophrenia is highly comorbid with other psychiatric disorders such as Alcohol Use Disorder (AUD) with a prevalence rate of 27% - 65%, which is significantly higher than AUD exhibited by the general population (6%). Research indicates that a higher rate of AUD in individuals suffering from schizophrenia may be related to the common neuronal pathways that underlie the expression of both disorders. The present study will determine whether the neonatal quinpirole (NQ) rodent model of schizophrenia will approximate the human condition and exhibit increased EtOH consumption. Rats will be treated neonatally with quinpirole or saline. Following the treatment period, rats will be tested for EtOH consumption using a 24-hour two-bottle free-access paradigm. The proposed research will test the hypothesis that rats neonatally treated with quinpirole will consume significantly greater amounts of EtOH than their saline counterparts.

No description available.