Ultrastructural and functional changes at the tripartite synapse during epileptogenesis in a model of temporal lobe epilepsy

Abstract

The persistent unresponsiveness of many of the acquired epilepsies to traditional antiseizure medications has motivated the search for prophylactic drug therapies that could reduce the incidence of epilepsy in this at risk population. These studies are based on the idea of a period of epileptogenesis that can follow a wide variety of brain injuries. Epileptogenesis is hypothesized to involve changes in the brain not initially associated with seizures, but which result finally in seizure prone networks. Understanding these changes will provide crucial clues for the development of prophylactic drugs. Using the repeated low-dose kainate rat model of epilepsy, we have studied the period of epileptogenesis following status epilepticus, verifying the latent period with continuous EEG monitoring. Focusing on ultrastructural properties of the tripartite synapse in the CA1 region of hippocampus we found increased astrocyte ensheathment around both the presynaptic and postsynaptic elements, reduced synaptic AMPA receptor subunit and perisynaptic astrocyte GLT-1 expression, and increased number of docked vesicles at the presynaptic terminal. These findings were associated with an increase in frequency of the mEPSCs observed in patch clamp recordings of CA1 pyramidal cells. The results suggest a complex set of changes, some of which have been associated with increasingly excitable networks such as increased vesicles and mEPSC frequency, and some associated with compensatory mechanisms, such as increased astrocyte ensheathment. The diversity of ultrastructural and electrophysiological changes observed during epileptogeneiss suggests that potential drug targets for this period should be broadened to include all components of the tripartite synapse.

Keywords: Astrocyte ensheathment; Electron microscopy; Epilepsy; Latent period; Patch clamp; Tripartite synapse; Ultrastructure; mEPSCs.

Figure 1:

KA-induced SE and video EEG recordings: A. Timeline showing the surgical placement of the electrode, the low-dose KA injections, and subsequent recording. B. Saline injections did not result in notable changes in power over the course of recording. Arrow indicates the time of injection. C. EEG traces exhibited no seizures D. Multiple KA injections were followed by an increase in power in the 0–25 Hz range. Spectral power of the EEG signal is increased for many hours following KA injections (arrow), consistent with the occurrence of multiple stage 4/5 seizures in this animal. The lines at 60 Hz reflects noise at this bandwidth in both recordings. Spectrograms (window = 30s, step = 30s) were generated using the Chronux Toolbox for Matlab (http://chronux.org). To account for the differences in scale between sham and KA injected animals, spectral power (arbitrary units) in each animal was normalized to 125% of the maximum log power observed in the first five hours. E. Voltage trace of an example seizure in a KA treated animal F. Voltage trace of spiking activity on day 6 of the latent period in a KA treated animal and corresponding spectrogram. G. Example trace on day 6 of a sham animal and corresponding spectrogram. The spectrogram in G uses the same scale color scale as in F so they can be directly compared.

Figure 2.

KA-induced SE induces disorganization of the CA1 stratum radiatum and neuronal cell death. It also leads to an increase in astrocyte cell body size and astrocyte process area. A. Semithin sections stained with toluidine blue in a sham animal and after KA injection. Boxed area represents the area of the stratum radiatum (SR) that was analyzed for the ultrastructural and morphometric analysis. The pyramidal cell (Py) layer after KA injection showed disorganization of pyramidal cell bodies which some appeared dark, indicating cell death. In SR the radial organization of apical dendrites of pyramidal cells is lost after KA injection. Scale bar: 20 um. B. Electron micrographs at low magnification showing the cell bodies of an astrocyte in CA1 stratum radiatum of both sham and KA injected rats. The astrocyte is false colored in blue to aid in visualization. Astrocyte after KA injection showed an increase in cell body area and thicker main processes. Scale bar: 0.1 um. C. Electron micrographs at high magnification showing the astrocytes enwrapping the synapse of CA3 Shaffer collateral on a CA1 spine (S) in the SR of sham and after KA injection. The perysinaptic astrocyte processes are false colored in blue to help visualization. Scale bar: 0.1 um

Figure 3.

KA-induced SE leads to an increase of astrocyte enwrapping around CA1 excitatory synapses. A. Single section electron micrographs of representative CA1 excitatory synapses within the stratum radiatum of a sham and KA injected animals. To aid visualization, the presynaptic terminal (T) in yellow and the astrocyte presynaptic process in blue. Arrowheads point out the edge of the PSD. Scale bar: 0.2 um B. Corresponding 3D-reconstructions from serial ultrathin sections from the spines shown in A. Scale cube is 0.2 um per side. C. Bar histogram showing the area of the presynaptic terminal and spine in shams and after KA injection. D. Bar histogram showing the percent of perisynaptic astrocyte enwrapping around the presynaptic terminal and the spine

Figure 4.

Postsynaptic densities are thinner and their number of docked synaptic vesicles increases after KA-induced SE. A. Schematic illustration of a CA1 synapse and their synaptic vesicle pool contained in an area up to 50 nm from the presynaptic plasma membrane. T: presynaptic terminal; S: spine. B. Bar histograms showing the area and thickness of the postsynaptic density (PSD) in shams and after KA injection. C. 3D-reconstructions of 2 examples of two PSDs (gray) and their docked synaptic vesicles (white) in shams and after KA injection. D. Average number of docked synaptic vesicles (DSvS) on 3D reconstructed PSDs reconstructed. Plots showing the correlation of DSVs with the PSD area.

Figure 5

Decrease of AMPAR subunit expression after KA-induced SE. A. Electron micrographs showing the post-embedding immunogold labeling for GluA1, GluA2 and GluA2/3 in the CA1 stratum radiatum of shams and after KA treatment. Most of the gold particles (10 nm in diameter) are observed at the postsynaptic density of the spines (s), some gold particles were observed extrasynaptically (cytoplasm and plasma membrane). Perisynaptic astrocyte (Ast) processes are observed around the spine. Scale bar: 0.2 um. B. Plots showing the average number and linear density of gold particles for GluA1, GluA2 and Glu2/3 at the synapse of shams and after KA injection.

Figure 6

Changes in mEPSCs during the latent period following KA-induced SE. A) Example traces showing mEPSCs from sham and KA-treated animals. B) The average frequency of mEPSCs is increased in experimental animals C) The average amplitude is not different between groups D) There is an increased number of large mEPSC in experimental animals during the latent period. While the average amplitude is not significantly different, the cumulative distributions are with a clear separation between the distributions that increases at the larger amplitudes.

Figure 7

Decrease in GLT-1 labeling at the astrocyte plasma membrane after KA-induced SE. A. Electron micrographs showing post-embedding immunogold labeling for GLT-1 (10 nm in diameter) on the plasma membrane and cytoplasm of perisynaptic astrocytes (Ast). S: spine; D: dendrite. Scale bar: 0.2 um. B. Plots showing the average number and density of gold particles for GLT-1 on the plasma membrane or in the cytosol of perisynaptic astrocytes.