Abstracts

Seizure-induced neuronal death is well-described, but less is known about non-lethal cellular sequelae of seizures on neurons. Previous clinical and animal studies suggest that seizures have pathological effects on dendritic spines, thus potentially altering normal synaptic function. However, most previous studies have only demonstrated long-term, chronic effects of seizures on spines using conventional fixed tissue analysis. Recent advances in cellular imaging technology allow high-resolution time-lapse imaging of dendritic spines in living tissue and have demonstrated rapid effects of physiological activity on spines on a time scale of minutes. Thus, we hypothesized that pathological seizure activity may also induce acute, rapid changes in dendritic spines. We have developed a method for directly imaging the effects of electrographic seizures on dendritic spines of neocortical neurons in intact mice [italic]in vivo[/italic]. Two-to-three month old green-fluorescent protein (GFP)-expressing trangenic mice (M-line, Washington University) were anesthetized with isoflurane and placed in a stereotaxic frame. A craniotomy was performed over frontal neocortex and a coverslip cemented over the craniotomy to form an imaging window. Wire EEG electrodes and a cannula for drug application were placed under the coverslip along the lateral aspect of the craniotomy and over an incision in the dura. A Zeiss LSM 510 Multiphoton microscope was used to image GFP-positive dendrites approximately 25-75 microns below the neocortical surface. We acquired Z-stacks of the same dendrites and associated spines every 15-30 minutes over three hours in control mice and mice infused with 4-aminopyridine locally below the cranial window to elicit electrographic seizures. We analyzed images off-line to assess changes in spine number (gain or loss) under control conditions and during/after seizures. Individual dendritic spines from neocortical dendrites in anesthetized mice [italic]in vivo[/italic] could be imaged repetitively over three hours with good resolution. Repetitive electrographic seizures could be induced following local application of 4-aminopyridine. In both control and seizure conditions, almost all existing spines remained stable over a three hour period, with loss of existing spines or formation of new spines occuring only rarely. Compared to controls, there was no significant effect of seizures on spine number. Surprisingly, seizures had no obvious, acute effects on dendritic spine number in the neocortex of anesthetized mice. Additional studies, involving more detailed analysis of spine morphology and motility, effects of anesthesia, longer-term chronic imaging, or other seizure models may reveal pathological effects of seizures on dendritic spines. (Supported by NIH 1 K02 NS045583-01)