Date of Award
Open Access Thesis
Medical Doctor (MD)
The purpose of this study was to create and optimize a model of mesial temporal lobe epilepsy through selective depletion of glutamine synthetase (GS) in the mouse hippocampus. Following validation of the model, preliminary studies attempted to characterize morphological astrocytic and synaptic changes that result from GS deficiency. Aim 1 established a novel mouse model of GS knockout in hippocampal astrocytes. Aim 2 tested whether localized hippocampal knockout of GS causes mice to exhibit an epilepsy-like phenotype. Aim 3 characterized the cellular effects of localized GS loss. To generate the knockout, Glul-floxed C57BL/6J mice were injected with four different adeno-associated viral vectors containing Cre-recombinase expression cassettes. Mice were also implanted with intracranial depth or screw recording electrodes and monitored for spontaneous seizures using 24-hour video-EEG recording for two weeks. To assess for provoked seizure sensitivity, seizures were induced with pentylenetetrazol (PTZ) prior to perfusion fixation. Brains were perfused, sectioned, and immunostained for analysis using standard and STED fluorescence microscopy. Knockout of GS, as evidenced by loss of GS immunoreactivity, was found over a greater area in brain regions injected with the AAV5 CMV and AAV8 GFAP serotypes. In addition, within each GS knockout region, AAV8 GFAP exhibited a significantly greater efficiency of knockdown compared to AAV5 CMV Legacy and AAV8 CMV (83.1% decreased fluorescence intensity, p=0.0003) and compared to AAV5 CMV (20.2% decreased fluorescence intensity, p=0.018). AAV8 GFAP exhibited near perfect target specificity (98.7% of GFP+ cells were astrocytes), while AAV5 CMV Legacy, AAV5 CMV, and AAV8 CMV targeted mostly neurons with varied degrees astrocyte labeling detected (10.0%, 21.3%, and 12.7% astrocytes, respectively. Sixty percent (3/5) of mice injected with AAV8 GFAP exhibited an epilepsy-like phenotype including spontaneous recurrent seizures that were clustered in the morning hours. Twenty-five percent (1/4) of control mice seized spontaneously over the same period. Additionally, focal GS knockout mice demonstrated significantly lower time to initial clonic twitch following PTZ administration compared to control mice (mean ± SEM: 41.2 ± 3.2 seconds vs. 65.83 ± 12.9 seconds, respectively; p=0.044). The effect on time to convulsive seizure was not statistically significant, though there was a trend of knockout animals proceeding to convulsions in less time (74.2 ± 9.4 seconds vs. 100.0 ± 18.0 seconds, p=0.20). Finally, examination of synaptic markers revealed decreased expression of PSD-95 surrounding GS- astrocytes compared to GS+ astrocytes, with sampled relative intensity of 0.57 ± 0.04 (p=0.002). Relative intensity (RI) of synaptophysin and gephyrin appeared to be unchanged in the sampled areas (synaptophysin RI 0.94 ± 0.15, p=0.87; gephyrin RI 0.94 ± 0.04, p=0.23). In this study, we created a novel model of mesial temporal lobe epilepsy by selectively knocking out GS in the hippocampal astrocytes of mice. Development of this monogenetic knockout model with effects restricted to the hippocampus and adjacent structures has the potential to more fully elucidate the impact of GS loss in this treatment-resistant disease. Initial examination of synaptic markers in GS depleted areas highlights the importance of glutamatergic synaptic transmission in epilepsy pathology.
Farina, Maxwell Gerard, "Localized Hippocampal Glutamine Synthetase Knockout: A Novel Model Of Mesial Temporal Lobe Epilepsy" (2019). Yale Medicine Thesis Digital Library. 3491.
This Article is Open Access