Date Thesis Awarded

5-2021

Access Type

Honors Thesis -- Access Restricted On-Campus Only

Degree Name

Bachelors of Science (BS)

Department

Applied Science

Advisor

Christopher A. Del Negro

Committee Members

Gregory D. Conradi Smith

Jennifer E. Bestman

Pamela S. Hunt

Abstract

The mesencephalic locomotor region is a command center in the midbrain responsible for regulating the excitation of reticulospinal neurons to facilitate locomotor movements. Previous studies have shown the pedunculopontine nucleus in the mesencephalic locomotor region is responsible for initiating the intensity and duration of locomotor behavior. These studies have specified that the glutamatergic neurons specifically in the pedunculopontine nucleus are responsible for its gating function. However, the underlying ion channel-level mechanisms that activate these neurons are not known, thus the central focus of this thesis. The pedunculopontine nucleus is involved in a longstanding theory about how locomotion initiates. According to the disinhibition hypothesis, at rest the motor pathway is tonically inhibited via the output layer of the basal ganglia, which prevents movement by default. An initiating signal that relieves the inhibition from basal ganglia to the mesencephalic locomotor region at the pedunculopontine nucleus can trigger bouts of locomotion. We hypothesized that initiation involves low threshold Ca2+ currents that generate low-threshold Ca2+ spikes and bursts upon release from inhibition. Our goal is to knockdown the mRNA for the gene that codes for CaV 3.1 ion channels that mediate low-threshold Ca2+ currents in glutamatergic neurons of the pedunculopontine nucleus to test our hypothesis, thus discovering the ionic mechanisms that help initiate locomotion. We were able to develop the methodology for the gene knockdown experiments. We show the process of localizing the pedunculopontine nucleus for stereotaxic injection of payload-carrying adenoviruses, and we recap the troubleshooting along the way. By finalizing the targeting coordinates for injection using GFP viral vector injections, we provide the experimental knowledge and framework to accurately and consistently hit the target region for gene knockdown. Nevertheless, because of 8-month shutdown of undergraduate research due to Covid-19, as of the writing on this thesis, the gene knockdown experiments remain incomplete. This thesis documents all the preparation, troubleshooting, and protocol fine-tuning to unravel the neural bases for locomotor program initiation at the gene and ion channel level of analysis.

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