Date of Award

2025-08-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Manuel Miranda-Arango

Abstract

Glycine functions as an inhibitory neurotransmitter in the central nervous system (CNS),and it is mainly involved in autonomic functions. The levels of extracellular glycine are regulated by two membrane transporters, glycine transporter 1 and glycine transporter 2 (GlyT1 and GlyT2, respectively). These transporters differ in function and localization within the CNS. For many years, studies suggested that only GlyT2 served as a neuronal marker; however, GlyT1 has been localized in neurons in recent studies. The expression of GlyT1 is widespread compared to GlyT2; hence, the main objective is to determine the nature of the expression of GlyT1 in areas devoid of GlyT2, especially in subcortical regions of the brain. More specifically, cell bodies labeled with Neuronal Nuclei (NeuN) antibody (Ab) in the subthalamic nuclei (STN) and substantia nigra (SN) have shown expression of GlyT1 at the plasma membrane. Staining with glial fibrillary acidic protein (GFAP) has demonstrated poor colocalization of GlyT1 in astrocytes. Furthermore, staining with Glutamate Decarboxylase 67 (GAD67) and GlyT1 has shown co- expression in groups of cell bodies and neuronal processes, suggesting a double-inhibitory phenotype. Nonetheless, to confirm these data, a knock-in mouse was generated to label GlyT1- positive cells with the reporter tdTomato (tdTom). Results have shown tdTom expression in neuronal cell bodies co-labeled with NeuN and GlyT1 antibodies (Ab's) in the STN and SN. Additional characterization of these cells includes the intracranial delivery of adeno-associated viral particles (AAVs) and expression of the reporter in glial or neuronal cells. Future directions also include the application of electrophysiological techniques to investigate the role of glycinergic circuits on inhibitory post-synaptic currents (IPSC) as well as behavioral studies to assess the in-vivo effect on basal ganglia-regulated functions by blockage of glycine receptors (GlyRs) in the GPe through intracranial infusions. Moreover, while analyzing GlyT1/tdTom expression across sections, the cerebellum engaged our interest due to its saturated expression. Staining with GAD67, showed co-expression with tdTom in Purkinje cells. This co-labeling suggests a double-inhibitory phenotype, as it is well- documented that Purkinje cells are GABAergic. Altogether, the project's hypotheses are supported by these data showing GlyT1 expression in neurons in subcortical brain structures and cerebellum.

Language

en

Provenance

Received from ProQuest

File Size

116 p.

File Format

application/pdf

Rights Holder

Rosa Atziri Perez

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