Date of Award

2025-08-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Manuel Miranda-Arango

Abstract

Glycine is one of the main inhibitory neurotransmitters in the central nervous system. Glycine is regulated by the glycine transporters 1 and 2 (GlyT1 and GlyT2, respectively) in the synaptic cleft. Both transporters participate in glycinergic inhibitory neurotransmission. Moreover, GlyT1 also participates in glutamatergic excitatory neurotransmission. GlyT2 is devoted to caudal regions of the brain and is used as a marker for glycinergic neurons. In contrast, GlyT1 is expressed across the brain in the rostrocaudal axis and is highly associated with GlyT1-positive glial cells. Data from our laboratory has identified GlyT1-positive neurons in the rodent brain. Therefore, we hypothesize that GlyT1-positive neurons are present in different regions across the CNS of rodents, and these regions use glycine to communicate. Three different aims were established to test this hypothesis and develop this new concept of having GlyT1-positive neurons in the rodent brain. The first includes using antibodies to detect GlyT1 and other markers for cell identification. The second aim is to identify glycinergic circuits within circuitries that are already established but have never been associated with glycine. The last aim involves exploring the DTR ablation cell system that has not been used in rats before. To do so, GlyT1-Cre knock-in line mice and rats were used to analyze the distributions of cells expressing the glycine transporter by labeling with Cre-dependent fluorescent reporters. Immunohistochemistry experiments were performed in the GlyT1-Cre dependent mouse line that expresses tdTomato under the promoter of GlyT1. Tissue was stained with various antibodies, such as glial fibrillary acidic protein (GFAP) and neuronal nuclear protein (NeuN), to identify GlyT1-positive astrocytes and neurons, respectively. GlyT1 and Glutamate Decarboxylase 67 (GAD67) antibodies were also used to identify GlyT1-positive neurons and GABAergic neurons. Tract-tracing studies were performed using the GlyT1-Cre dependent mice and rats. Adeno-Associated Virus (AAV) particles were delivered in different brain regions to assess their GlyT1-positive projections. Finally, a pilot study using the DTR system was performed to evaluate the role of striatal GlyT1-positive neurons in voluntary motor control. Results from these experiments showed GlyT1-positive neurons and glial cells in the striatum, globus pallidus external and internal segments (GPe and GPi), hippocampus subregions (CA1, CA3, dentate gyrus (DG)), and reticular nucleus of the thalamus (RT). Tract-tracing experiments demonstrated GlyT1-positive anatomical connections from the lateral hypothalamic area (LHA), RT, zona incerta (ZI), and hippocampus to other forebrain regions. Results from the DRT system provided the basis for an optimization of the experimental design in rats, a rodent model not tested in published work. Overall, results from this project provide the identification of cells and projection patterns of GlyT1-positive neurons in different brain regions. Future directions include mapping the expression patterns into a high-resolution atlas such as the Allen Reference Atlas (ARA) and the Brain Maps 4.0 (BM 4.0) for mouse and rat, respectively. More restrictive AVV injections will be performed in the different brain areas of interest, and their projection patterns mapped to the previously mentioned atlases. Changes to the DTR experimental design will be made to evaluate the role of GlyT1-positive neurons in the striatum during behavioral assays that will test their contributions to voluntary motor control. Altogether, this study characterizes GlyT1 cells' phenotypes and projections across the rodent CNS.

Language

en

Provenance

Received from ProQuest

File Size

157 p.

File Format

application/pdf

Rights Holder

Laura Patricia Montes

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