Regionally localized recurrent excitation in the dentate gyrus of a cortical contusion model of posttraumatic epilepsy

Abstract

Posttraumatic epilepsy is a frequent consequence of brain trauma, but relatively little is known about how neuronal circuits are chronically altered after closed head injury. We examined whether local recurrent excitatory synaptic connections form between dentate granule cells in mice 8-12 wk after cortical contusion injury. Mice were monitored for behavioral seizures shortly after brain injury and < or = 10 wk postinjury. Injury-induced seizures were observed in 15% of mice, and spontaneous seizures were observed weeks later in 40% of mice. Timm's staining revealed mossy fiber sprouting into the inner molecular layer of the dorsal dentate gyrus ipsilateral to the injury in 95% of mice but not contralateral to the injury or in uninjured controls. Whole cell patch-clamp recordings were made from granule cells in isolated hippocampal brain slices. Cells in slices with posttraumatic mossy fiber sprouting had an increased excitatory postsynaptic current (EPSC) frequency compared with cells in slices without sprouting from injured and control animals (P < 0.001). When perfused with Mg(2+)-free artificial cerebrospinal fluid containing 100 microM picrotoxin, these cells had spontaneous bursts of EPSCs and action potentials. Focal glutamate photostimulation of the granule cell layer evoked a burst of EPSCs and action potentials indicative of recurrent excitatory connections in granule cells of slices with mossy fiber sprouting. In granule cells of slices without sprouting from injured animals and controls, spontaneous or photostimulation-evoked epileptiform activity was never observed. These results suggest that a new regionally localized excitatory network forms between dentate granule cells near the injury site within weeks after cortical contusion head injury.

Fig. 1.

Mice develop injury-induced and spontaneous seizures after severe controlled cortical impact (CCI) injury. Cumulative probability plot of the 1st observed seizure after CCI injury (time 0). Seizure counts were reset after 1 and 7 day to separate immediate, early, and spontaneous seizures.

Fig. 2.

Cavitation into the hippocampus and posttraumatic mossy fiber sprouting 8–12 wk after severe CCI injury. A: representative Nissl stain of the dentate gyrus contralateral to the injury site. B: Timm's stain of the same section in A shows the absence of mossy fiber sprouting in the inner molecular layer. C, E, and G: representative Nissl-stained images of the ipsilateral dentate gyrus at the injury site. →, indicate severe thinning of the granule cell layer. D, F, and H: Timm's stain of the same sections in C, E, and G. Note the presence of moderate mossy fiber sprouting in all sections (→). Percentage of mice with each type of lesion/Timm stain at the injury site is indicated. Scale bar is 100 μm.

Fig. 3.

Increased spontaneous excitatory postsynpatic currents (sEPSCs) in slices of the ipsilateral dentate gyrus with mossy fiber sprouting (MFS). A–D: representative whole cell patch-clamp recordings of granule cells in slices from control (A), contralateral (B), and ipsilateral (C) without MFS and ipsilateral with MFS (D). Boxed areas of each trace are enlarged below. E: average EPSC frequency for cells in each treatment group. The numbers of cells are indicated in parentheses above each bar. F: EPSC frequency plotted as a function of Timm score. Solid lines, mean EPSC values; dotted lines, ±S D. G: average amplitude for cells in each of the 4 treatment groups. Error bars indicate mean ± SD. Asterisk, P < 0.001.

Fig. 4.

Spontaneous bursts of compound EPSCs in cells from slices with MFS ipsilateral to the injury. Representative whole cell voltage-clamp recordings of granule cells in a slice from an uninjured control mouse (A), a contralateral slice in an injured mouse (B), a slice ipsilateral to the injury without MFS (C), and a slice ipsilateral to the injury with MFS (D). →, expanded portion of the trace in D. Slices were incubated with Mg²⁺-free artificial cerebrospinal fluid (ACSF) and 100 μM PTX; V_m = −70 mV for all recordings.

Fig. 5.

Spontaneous epileptiform bursts of action potentials in cells of slices with MFS ipsilateral to the injury. Representative whole cell current-clamp recordings of granule cells in a slice from an uninjured control mouse (A), a contralateral slice in an injured mouse (B), a slice ipsilateral to the injury without MFS (C), and a slice ipsilateral to the injury with MFS (D). Inset: expanded sections of the underlined portions of the trace in D labeled 1 and 2. Resting membrane potential is indicated for each trace.

Fig. 6.

Granule cell—granule cell connections are not detected by glutamate photostimulation in cells from slices contralateral to the injury. A: voltage-clamp recordings at −70 mV from a granule cell. B: current-clamp recordings at resting membrane potential from the same cell as A. Dotted vertical lines, the time of stimulation. Numbers to the left of each trace indicate corresponding stimulus position shown in C. C: Nissl-stained image of the slice from which the recorded cell was obtained. Numbers correspond to the approximate locations along the granule cell layer that photostimulation was applied to give the numbered responses recorded in A and B. Stimulation position 3 (circled) is the approximate location of the recorded cell. Note that direct activation of the recorded cell induced an inward current and burst of action potentials. D: Timm's stain image of the same section in C indicating no MFS into the inner molecular layer.

Fig. 7.

Granule cell—granule cell connections are detected by glutamate photostimulation in cells from slices ipsilateral to the injury with MFS. A: voltage-clamp recordings at −70 mV from a granule cell. B: current-clamp recordings at resting membrane potential from the same cell as A. Dotted vertical lines, the time of stimulation. C: Nissl-stained image of the slice from which the recorded cell was obtained. Numbers correspond to the approximate location along the granule cell layer that photostimulation was applied to give the responses recorded in A and B. Stimulation site number 2 (circled) is the approximate location of the recorded cell. Note that direct photoactivation of the recorded cell induced an inward current and burst of action potentials (A2 and B2). Activity induced in neurons at locations 3 and 4 resulted in synaptic responses (A3 and A4) and action potentials (B3 and B4) in the recorded granule cell. D: Timm's stain image of the same section in C. Note: MFS surrounds the position of the recorded cell.

Fig. 8.

Variation in evoked EPSC (eEPSC) responses in granule cells from slices ipsilateral to the lesion. A–C: representative responses in a single neuron at 3 different stimulation locations in the granule cell layer. Each response shows 5 consecutive overlapping responses with 3 consecutive individual traces separately shown below. A: photostimulation did not evoke a response. B: a mild response of 1–2 EPSCs was consistently evoked in each of 5 trials. C: a more robust response that consisted of 5–9 eEPSCs in each of 5 trials after photostimulation at a different site in the granule cell layer. D: number of EPSCs per 100 ms before and after stimulation for each representative response in A–C. Contralateral responses are averaged across all stimulation locations in 5 neurons. →, the time of stimulation. E: frequency histogram shows the distribution of the average number of eEPSCs at 18 stimulation sites that had a positive response.

Fig. 9.

eEPSPs and action potentials in granule cells ipsilateral to CCI injury after photostimulation at distant locations in the granule cell layer. A–C: representative responses in a single neuron at 3 different stimulation locations of the granule cell layer. Each response shows 5 consecutive overlapping responses. A: no response evoked by photostimulation. B: a mild response was evoked with 1 of 5 responses reaching action potential threshold. Photostimulation at this location evoked an average of 3 EPSCs in voltage-clamp mode. C: a more robust evoked response that consisted of a small burst of action potentials in each of 5 trials. Photostimulation at this location evoked an average of 10 EPSCs in voltage-clamp mode. Insets show expanded portion of one trace. →, the time of stimulation.