Acetylcholine modulates neuronal activity in the brain with different responses in activity depending on the region of the brain. Our study was focused on the cholinergic modulation of excitatory synaptic transmission in the monkey anterior cingulate cortex (ACC) and lateral prefrontal cortex (LPFC), with specific focus on the effects of carbachol, a cholinergic agonist, on spontaneous excitatory postsynaptic currents (sEPSCs) and on the expression the muscarinic cholinergic type II (M2) receptor in these regions. We used electrophysiology to analyze the effects of carbachol on sEPSC of layer 3 (LIII) pyramidal neurons from each area. We used confocal microscopy to study the M2 colocalization with axon terminals labeled with vesicular glutamate transporter 1 (VGLUT1) in the ACC and LPFC, and the colocalization of M2 with specific axon terminals from the amygdala labeled with tracer and terminating in the ACC. Results from the electrophysiological experiments showed that both the ACC and LPFC L3 neurons responded to carbachol by decreasing the frequency of sEPSCs. Cells from the LPFC showed a decrease in sEPSC frequency after 4 minutes in carbachol, an earlier timepoint than ACC neurons, which showed a decrease in sEPSCs frequency after 6 minutes in carbachol. In the confocal studies, M2 expression and colocalization with VGLUT1 terminals in the ACC and LPFC were observed. However, we observed a greater total area of M2 expression in the ACC versus the LPFC in layer 1. We found minimal colocalization of the M2 receptor with axon terminals from the amygdala in the ACC. Together, our data show that acetylcholine has distinct interactions with neurons and pathways in ACC and LPFC, which may be related to the distinct function of the two areas in cognition, learning and memory.

No description available.

abstract: Older adults often experience communication difficulties, including poorer comprehension of auditory speech when it contains complex sentence structures or occurs in noisy environments. Previous work has linked cognitive abilities and the engagement of domain-general cognitive resources, such as the cingulo-opercular and frontoparietal brain networks, in response to challenging speech. However, the degree to which these networks can support comprehension remains unclear. Furthermore, how hearing loss may be related to the cognitive resources recruited during challenging speech comprehension is unknown. This dissertation investigated how hearing, cognitive performance, and functional brain networks contribute to challenging auditory speech comprehension in older adults. Experiment 1 characterized how age and hearing loss modulate resting-state functional connectivity between Heschl’s gyrus and several sensory and cognitive brain networks. The results indicate that older adults exhibit decreased functional connectivity between Heschl’s gyrus and sensory and attention networks compared to younger adults. Within older adults, greater hearing loss was associated with increased functional connectivity between right Heschl’s gyrus and the cingulo-opercular and language networks. Experiments 2 and 3 investigated how hearing, working memory, attentional control, and fMRI measures predict comprehension of complex sentence structures and speech in noisy environments. Experiment 2 utilized resting-state functional magnetic resonance imaging (fMRI) and behavioral measures of working memory and attentional control. Experiment 3 used activation-based fMRI to examine the brain regions recruited in response to sentences with both complex structures and in noisy background environments as a function of hearing and cognitive abilities. The results suggest that working memory abilities and the functionality of the frontoparietal and language networks support the comprehension of speech in multi-speaker environments. Conversely, attentional control and the cingulo-opercular network were shown to support comprehension of complex sentence structures. Hearing loss was shown to decrease activation within right Heschl’s gyrus in response to all sentence conditions and increase activation within frontoparietal and cingulo-opercular regions. Hearing loss also was associated with poorer sentence comprehension in energetic, but not informational, masking. Together, these three experiments identify the unique contributions of cognition and brain networks that support challenging auditory speech comprehension in older adults, further probing how hearing loss affects these relationships. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2019

abstract: Medulloblastoma is the most common malignant pediatric brain cancer and is classified into four different subgroups based on genetic profiling: sonic hedgehog (SHH), WNT, Group 3 and 4. Changes in gene expression often alter the progression and development of cancers. One way to control gene expression is through the acetylation and deacetylation of histones. More specifically in medulloblastoma SHH and Group 3, there is an increased deacetylation, and histone deacetylase inhibitors (HDACi) can be used to target this change. Not only can HDACi target increases in deacetylation, they are also known to induce cell cycle arrest and apoptosis. The combination of these factors has made HDACi a promising cancer therapeutic. Panobinostat, a hydrophobic, small molecule HDACi was recently identified as a potent molecule of interest for the treatment of medulloblastoma. Furthermore, panobinostat has already been FDA approved for treatment in multiple myeloma and is being explored in clinical trials against various solid tumors. The laboratory is interested in developing strategies to encapsulate panobinostat within nanoparticles composed of the biodegradable and biocompatible polymer poly(lactic acid)-poly(ethylene glycol) (PLA-PEG). Nanoparticles are formed by single emulsion, a process in which hydrophobic drugs can be trapped within the hydrophobic nanoparticle core. The goal was to determine if the molecular weight of the hydrophobic portion of the polymer, PLA, has an impact on loading of panobinostat in PLA-PEG nanoparticles. Nanoparticles formulated with PLA of varying molecular weight were characterized for loading, size, zeta potential, controlled release, and in vivo tolerability. The results of this work demonstrate that panobinostat loaded nanoparticles are optimally formulated with a 20:5kDa PLA-PEG, enabling loading of ~3.2 % w/w panobinostat within nanoparticles possessing an average diameter of 102 nm and surface charge of -8.04 mV. Panobinostat was released from nanoparticles in a potentially biphasic fashion over 72 hours. Nanoparticles were well tolerated by intrathecal injection, although a cell culture assay suggesting reduced bioactivity of encapsulated drug warrants further study. These experiments demonstrate that the molecular weight of PLA influences loading of panobinostat into PLA-PEG nanoparticles and provide basic characterization of nanoparticle properties to enable future in vivo evaluation. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2019

Pas de résumé / No abstract

My research is designed to test the hypothesis that a single nucleotide polymorphism (SNPs) in the non-coding region of the GCH1 gene suppresses GCH1 mRNA translation and generates a novel peptide, and that both of these phenomena contribute to downregulation of the dopamine synthesis pathways. The GCH1 gene codes for the protein guanosine triphosphate (GTP) cyclohydrolase 1 (GCH1), a key regulator of dopamine synthesis. I focused on a SNP resulting from substitution of a cytosine (C) with thymine (T) at location +142 (position from the transcription start site) in the 5’ untranslated region (UTR) of the GCH1 gene (+142C>T SNP), because it is associated with a heritable movement disorder and subtype of dystonia called DYT5. Thus, my research is designed to investigate molecular mechanisms associated with the pathogenesis of DYT5. To test my hypothesis, I used an in vitro model in which human embryonic kidney (HEK) 293T cells were transfected with cDNA for wild type GCH1, GCH1 containing the +142C>T SNP sequence, or control sequences. Firefly luciferase (Fluc) reporter assays showed that the +142C>T SNP introduces an upstream open reading frame (uORF), which suppresses translation of GCH1. The reduced GCH1 translation fits well with the reduction in GCH1 protein and dopamine reported in DYT5. The uORF introduced by the +142C>T SNP is predicted to synthesize a 73 amino acid novel peptide, which I have named DRDp73. As a first step toward understanding the role of DRDp73 cellular function, I performed a bioinformatics analysis. This analysis predicted existence of a nuclear localization signal, a large “disordered” region and a nucleic acid binding alpha-helical domain in DRDp73 revealing interesting clues to DRDp73’s potential biological function. To test predictions provided by the bioinformatics analysis, I characterized the expression, subcellular localization and the impact of DRDp73 on cell viability. I used a custom-made DRDp73 antibody, and a commercially available GCH1 antibody to detect both of the proteins. I detected DRDp73 and GCH1 in HEK293T cells overexpressing the +142T GCH1 (GCH1 in DYT5) plasmid. However, GCH1 expression in these cells was significantly lower than that in HEK293T cells transfected with wildtype GCH1 plasmid. Since HEK293T cells divide rapidly, I predicted that DRDp73 is degraded and cleared during cellular division. Therefore, in additional studies, I inhibited the proteasome and autophagy pathways, key regulators of protein degradation. Under these circumstances, DRDp73 was localized to the nucleus, consistent with the predicted nuclear localization signal revealed by bioinformatics, and GCH1 was localized to the cytoplasm. In addition, viability of the HEK293T cells expressing DRDp73 was compromised. These findings confirmed that the +142C>T SNP is associated with generation of DRDp73, downregulation of GCH1 expression, and provided evidence that DRDp73 accumulation may adversely affect cell viability. The HEK293T cells do not synthesize dopamine, the neurotransmitter involved in motor function. Moreover, since the HEK293T cells divide rapidly, analysis of the long term effects of protein accumulation becomes difficult. Therefore, I used human origin SK-N-BE(2)-M17 (a twice sub-cloned cell line from the SK-N-BE (2) neuroblastoma cell line) and SH-SY5Y cells (third sub-clone of SK-N-SH neuroblastoma cell line). Both cell lines differentiate into non-dividing cells, express GCH1 and neuronal markers upon differentiation, and are excellent models of dopaminergic cells, such as the midbrain dopaminergic neurons affected in DYT5 dystonia. My data from SK-N-BE(2)-M17 and SH-SY5Y cells show that endogenous GCH1 protein is localized to the nucleus, cytoplasm and processes. In both types of cells transfected with the +142T GCH1 plasmid, DRDp73 also localizes to the nucleus, cytoplasm and processes. GCH1 expression is reduced in the nucleus in both neuroblastoma cells expressing DRDp73, and is found predominantly in the cytoplasm and processes. The dopamine biosynthetic pathway is compromised in DRDp73 expressing SK-N-BE(2)-M17 cells. Thus, the +142C>T SNP downregulated GCH1 expression and compromised dopamine biosynthesis in the neuronal-like cells. In the final set of studies, I analyzed GCH1 expression in lymphoblastoid cells from DYT5 patients. GCH1 expression was reduced in the patient-derived cells compared to that in cells from non-DYT5 individuals. In summary, my work identified dual consequences of DYT5-associated +142C>T SNP in the non-coding region of the GCH1 gene: Suppression of GCH1 translation, and production of the novel DRDp73 peptide. DRDp73 is a novel biomarker for DYT5, and the DRDp73 antibody is a potential novel diagnostic tool. My results expand current understanding of the SNP-uORF encoded proteins, and advances the field of SNP-uORF associated diseases by offering novel mechanistic and functional insights into SNP-uORF encoded peptides. My work also identifies future studies, for example using DYT5 patient derived cells, primary neuronal cultures and animal models that could significantly advance this emerging biomedical field. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the Doctor of Philosophy. / Summer Semester 2017. / July 19, 2017. / dopamine, dystonia, gene, regulation, SNP, uORF / Includes bibliographical references. / Pradeep G. Bhide, Professor Directing Dissertation; Debra Ann Fadool, University Representative; Richard S. Nowakowski, Committee Member; Heather Flynn, Committee Member; Akash Gunjan, Committee Member.

<p>Neurotrophic factors and their receptors are crucial for the formation of neuronal connections and for the neuronal survival during embryonic development of the nervous system. This has particularly been shown by studies of the PNS. The work of this thesis has aimed at clarifying where and when in the developing retina and its tectal target (as parts of the CNS), certain neurotrophic factors come into play. In a functional perspective, the focus has been on the possible involvement of these factors in the regulation of cell death / survival. Therefore, naturally occuring cell death in the developing avian retina was first studied. From the results, it is concluded that there is an early and a late phase of cell death in the developing retina. The cells dying during the early phase are proliferating retinal precursor cells, and the cells dying later are in the process of terminal differentiation. The timing and distribution of the dying cells suggest that the cell death is regulated. During retinal development two retinal cell types, amacrine cells and horizontal cells, express TrkA, a receptor for the neurotrophin nerve growth factor (NGF). These amacrine cells are shown to undergo cell death during the late cell death phase, but can be rescued by exogenous NGF. The horizontal cells in turn, are supported by NGF in an autocrine manner, and therefore survive. Two other neurotrophic factors brain-derived neurotrophic factor (BDNF) and glial cell-line derived neurotrophic factor (GDNF) are both expressed in the retina and in the target for retinal ganglion cells, the optic tectum. By the results, it is concluded that neuronal activity can regulate the expression of BDNF in the retina and optic tectum. Furthermore, GDNF can stimulate neurite outgrowth from retinal explants of a certain age. Taken together, the main result of this thesis is that neurotrophic factors indeed can work as autocrine survival factors in the developing CNS. </p>

<p>Members of the transforming growth factor-β (TGF-β) superfamily exhibit a range of effects on a host of different cell types. They signal through heteromeric complexes of serine/threonine kinase receptors of type I and type II. Gene targeted mutations of both factors and receptors have revealed that many of them are involved in early embryonic development. This thesis examines the receptors for this superfamily in the nervous system, especially bone morphogenetic protein receptor type II (BMPR-II). It was cloned from chicken nervous tissue, and its and other receptors’ expression in peripheral ganglia, spinal cord and brain of chicken, rat and mouse were examined. BMPR-II, ActR-II and ActR-IA were abundantly expressed throughout development in the nervous system, however with temporal regulation. One ligand of BMPR-II, BMP-7, was found to act synergistically with NT-3 and GDNF on subsets of peripheral neurons to promote survival and neurite outgrowth. A knock-in mouse was generated, encoding a truncated form of BMPR-II coupled to the endogenous tyrosine hydroxylase (TH) gene with an internal ribosome entry site (IRES). For ES-cell selection, a neomycin resistance gene was incorporated into the construct. Homozygous mice carrying the knock-in allele exhibited a small, hypokinetic phenotype. Levels of dopamine, noradrenaline and serotonin were measured, and the catecholamines were found to be lowered, dopamine as much as 97% in the caudate nucleus. The low catecholamine levels may not be an effect of the truncated BMPR-II, but rather a consequence of the knock-in construct reducing TH transcriptional rate. The TH hypomorphic mouse strain generated could find use as a model for catecholamine impaired systems, as seen in Parkinson’s disease.</p>

<p>Stress incontinence and overactive bladder are disorders with a common symptom, urinary incontinence, which is a serious medical and social handicap. Several neurotransmitters regulate the function of the lower urinary tract, including noradrenaline, acetylcholine, and serotonin.</p><p>The present study is part of the search for pharmacological incontinence drugs. The aims of this thesis were to improve the existing pharmacological treatments of urinary incontinence and to look for alternative treatments: i) an α₁-adrenergic agonist that preferentially affects urethral over blood pressure was tested <i>in vivo</i>; ii) a modified cystometry model was developed for screening of muscarinic antagonists, by construction of a complete dose-response curve in each individual animal; iii) a new muscarinic antagonist, PNU-171990, was pharmacologically characterized <i>in vitro</i> and <i>in vivo</i>; iv) functional differences of the isomers of the muscarinic agonist BM-5 were characterized in the urinary bladder and ileum, <i>in vitro</i> and <i>in vivo</i>; v) the role of serotonin 5-HT_2A, 5-HT₃ and 5-HT₄ receptors were characterized on urinary bladder contractions <i>in vivo</i>.</p><p>In the search for urethra selective compounds, the α₁-adrenoceptors agonists phenylephrine and phenylpropanolamine selectively enhanced blood pressure as compared to the urethral pressure in rabbit. This is in contrast to the effect of oxymetazoline and NS-49. Muscarinic antagonists produced a dose-dependent inhibition of the volume-induced micturition pressure in the rat. PNU-171990, a non-selective muscarinic antagonist, revealed selectivity for urinary bladder pressure over salivation (P<0.05). (R)-BM-5 induced bladder contraction and saliva secretion in cats. The selective serotonin 5-HT_2A and 5-HT₃ receptor antagonists, ketanserin and tropisetron, both inhibited the effect of chemically induced bladder contraction in the anaesthetized cat.</p><p>In conclusion, an urethral-selective α_1A-adrenoceptor agonist may be a good treatment of stress incontinence. A bladder-selective competitive muscarinic antagonist is considered a good pharmacotherapy for overactive bladder. In addition, the 5-HT_2A and 5-HT₃receptor antagonist may improve lower urinary tract symptoms.</p>

Thesis (Ph. D.)--University of Chicago, Committee on Conceptual and Historical Studies of Science, June 2001. / Includes bibliographical references. Also available on the Internet.