Date of Award
Doctor of Philosophy (PhD)
Glutamatergic signaling is fundamental to human behavior and its dysfunction is implicated in a wide range of neurological and neurodegenerative disorders. Excitatory amino-acid transporters (EAATs) – of which four primary subtypes (EAAT1, EAAT2, EAAT3, and EAAT4) exist in the human brain – are primary regulators of glutamate signaling. Endogenously-expressed at either low (EAAT4-low) or high (EAAT4-high) concentration in cerebellar Purkinje cells, EAAT4 has been shown to regulate Purkinje cell activity in the cerebellar molecular layer. However, differences in glutamatergic signaling at the circuit and single-synaptic levels in EAAT4-low and EAAT4-high regions are poorly understood. To characterize EAAT4-specific effects on excitatory circuits in the cerebellum, we microinjected the glutamate sensor iGluSnFR (SnFR) into the cerebellar molecular layer of mice and examined SnFR activity under basal and evoked conditions in cerebellar slices. SnFR measurements indicated that glutamate may decay more rapidly in EAAT4-high regions and that glutamate may decay more slowly in EAAT4-low regions. Computerized simulations mimicking cerebellum-like environments supported this theory and provided further nuance to EAAT4-dependent effects on excitatory circuits, suggesting that EAAT4-high and EAAT4-low regions have distinct glutamate receptor signaling patterns. Overall, these findings are the first: 1) To use computer simulations to accurately predict functional outcomes of EAAT4/glutamate interactions in the cerebellum and 2) To describe of how changes in EAAT4 concentration may affect excitatory circuits in the cerebellum at single synapses. EAAT4 has been shown to modulate astrocytic glutamate in the cerebellum. However, how EAAT4-low and EAAT4-high regions differentially respond to astrocytic glutamate release modality (slow or fast) is unknown. Through the theoretical examination of glutamate behavior and glutamate receptor kinetics in computer simulations in EAAT4-low and EAAT4-high conditions, EAAT4-high models were shown to be less sensitive to the effects of both slow and fast astrocytic glutamate release on glutamate levels and receptor conductance, suggesting a primary regulatory role for EAAT4 regarding astrocytic glutamate. Overall, these results provide insight not only into the impact of EAAT subtype on excitatory signaling in the cerebellum but the groundwork for a deeper understanding of EAAT4’s therapeutic value in alleviating the effects of neurological and neurodegenerative conditions.