Altered dendritic distribution of EPS8 after in vitro seizure-like activity in rat hippocampal neurons
Location
Philadelphia, PA
Start Date
30-4-2025 1:00 PM
End Date
30-4-2025 4:00 PM
Description
INTRODUCTION: Early-life seizures (ELS) often precede the onset of cognitive comorbidities. Prior studies have linked cognitive disorders with altered structural plasticity, the neuronal activity-dependent changes in neuronal structure. Specifically, plasticity changes linked to cognitive deficits are often at the level of dendritic spines, small protrusions off the dendrite that are the postsynaptic sites of most excitatory synapses in the brain. However, the underlying molecular mechanisms linking seizures to structural plasticity changes are not fully understood.
OBJECTIVES: The primary objective of this study was to determine if in vitro seizure-like activity in hippocampal neurons changes the distribution of epidermal growth factor receptor pathway substrate 8 (EPS8), an actin-capping protein that regulates the polymerization of the dendritic spine actin cytoskeleton, and therefore dendritic spine morphology. We have previously shown dendritic spine structural plasticity changes after induction of seizure-like activity in day in vitro (DIV17) neurons, and that spine density changes were dependent on when seizure-like activity was induced. We hypothesized that inducing seizure-like activity in rat hippocampal neurons would change distribution of EPS8 within the dendrite and dendritic spine, and that the EPS8 changes would correlate with spine density and structural plasticity change.
METHODS: We induced seizure-like activity in cultured rat hippocampal neurons with a zero Mg2+ solution at DIV10, 14, or 17. Cells were transfected with CellLight™ Actin-GFP, BacMam 2.0 (Invitrogen) to label actin, then fixed 1 hour, 3 days, or 7 days after seizure-like activity (DIV10+0, +3, or +7, for example). EPS8 was detected in fixed cells using immunocytochemistry. Labeling was imaged with a confocal microscope, then images were analyzed in ImageJ to determine EPS8 concentration ratio of dendritic spine:shaft in neurons exposed to zero Mg2+ compared to untreated controls by t-test.
RESULTS: Our preliminary results show that the EPS8/GFP spine:shaft ratio of DIV10 neurons decreases in zero Mg2+ groups when compared to control groups immediately after seizure-like activity, representing a decreased EPS8 protein concentration at dendritic spines compared to the dendritic shaft. The EPS8 was more evenly distributed throughout the dendrite in contrast to control groups, which exhibit a preference for EPS8 accumulation in the dendritic spines. EPS8/GFP spine:shaft ratio rebounds within 3 days, by DIV10+3. The EPS8/GFP spine:shaft ratio remains elevated through day 7 (DIV10+7). We did not see a change in EPS8/GFP spine:shaft ratio when seizure-like activity was induced at DIV14. Interestingly, three days after seizure-like activity was induced at DIV17 (DIV17+3), a time point at which we previously showed an inability to induce dendritic spine structural plasticity, we found a significant decrease in EPS8/GFP spine:shaft ratio compared to control neurons.
CONCLUSION: These results suggest that early seizure-like activity alters the timeline of EPS8 dispersion throughout dendritic spines, potentially contributing to seizure-activity induced changes in dendritic spine structural plasticity and morphology.
Embargo Period
5-20-2025
Altered dendritic distribution of EPS8 after in vitro seizure-like activity in rat hippocampal neurons
Philadelphia, PA
INTRODUCTION: Early-life seizures (ELS) often precede the onset of cognitive comorbidities. Prior studies have linked cognitive disorders with altered structural plasticity, the neuronal activity-dependent changes in neuronal structure. Specifically, plasticity changes linked to cognitive deficits are often at the level of dendritic spines, small protrusions off the dendrite that are the postsynaptic sites of most excitatory synapses in the brain. However, the underlying molecular mechanisms linking seizures to structural plasticity changes are not fully understood.
OBJECTIVES: The primary objective of this study was to determine if in vitro seizure-like activity in hippocampal neurons changes the distribution of epidermal growth factor receptor pathway substrate 8 (EPS8), an actin-capping protein that regulates the polymerization of the dendritic spine actin cytoskeleton, and therefore dendritic spine morphology. We have previously shown dendritic spine structural plasticity changes after induction of seizure-like activity in day in vitro (DIV17) neurons, and that spine density changes were dependent on when seizure-like activity was induced. We hypothesized that inducing seizure-like activity in rat hippocampal neurons would change distribution of EPS8 within the dendrite and dendritic spine, and that the EPS8 changes would correlate with spine density and structural plasticity change.
METHODS: We induced seizure-like activity in cultured rat hippocampal neurons with a zero Mg2+ solution at DIV10, 14, or 17. Cells were transfected with CellLight™ Actin-GFP, BacMam 2.0 (Invitrogen) to label actin, then fixed 1 hour, 3 days, or 7 days after seizure-like activity (DIV10+0, +3, or +7, for example). EPS8 was detected in fixed cells using immunocytochemistry. Labeling was imaged with a confocal microscope, then images were analyzed in ImageJ to determine EPS8 concentration ratio of dendritic spine:shaft in neurons exposed to zero Mg2+ compared to untreated controls by t-test.
RESULTS: Our preliminary results show that the EPS8/GFP spine:shaft ratio of DIV10 neurons decreases in zero Mg2+ groups when compared to control groups immediately after seizure-like activity, representing a decreased EPS8 protein concentration at dendritic spines compared to the dendritic shaft. The EPS8 was more evenly distributed throughout the dendrite in contrast to control groups, which exhibit a preference for EPS8 accumulation in the dendritic spines. EPS8/GFP spine:shaft ratio rebounds within 3 days, by DIV10+3. The EPS8/GFP spine:shaft ratio remains elevated through day 7 (DIV10+7). We did not see a change in EPS8/GFP spine:shaft ratio when seizure-like activity was induced at DIV14. Interestingly, three days after seizure-like activity was induced at DIV17 (DIV17+3), a time point at which we previously showed an inability to induce dendritic spine structural plasticity, we found a significant decrease in EPS8/GFP spine:shaft ratio compared to control neurons.
CONCLUSION: These results suggest that early seizure-like activity alters the timeline of EPS8 dispersion throughout dendritic spines, potentially contributing to seizure-activity induced changes in dendritic spine structural plasticity and morphology.