Removing entorhinal cortex input to the dentate gyrus does not impede low frequency oscillations, an EEG-biomarker of hippocampal epileptogenesis

Abstract

Following prolonged perforant pathway stimulation (PPS) in rats, a seizure-free "latent period" is observed that lasts around 3 weeks. During this time, aberrant neuronal activity occurs, which has been hypothesized to contribute to the generation of an "epileptic" network. This study was designed to 1) examine the pathological network activity that occurs in the dentate gyrus during the latent period, and 2) determine whether suppressing this activity by removing the main input to the dentate gyrus could stop or prolong epileptogenesis. Immediately following PPS, continuous video-EEG monitoring was used to record spontaneous neuronal activity and detect seizures. During the latent period, low frequency oscillations (LFOs), occurring at a rate of approximately 1 Hz, were detected in the dentate gyrus of all rats that developed epilepsy. LFO incidence was apparently random, but often decreased in the hour preceding a spontaneous seizure. Bilateral transection of the perforant pathway did not impact the incidence of hippocampal LFOs, the latency to epilepsy, or hippocampal neuropathology. Our main findings are: 1) LFOs are a reliable biomarker of hippocampal epileptogenesis, and 2) removing entorhinal cortex input to the hippocampus neither reduces the occurrence of LFOs nor has a demonstrable antiepileptogenic effect.

Figure 1. Spontaneous electrographic events recorded from the dentate gyrus in a freely-moving rat during the latent period following 8 hours of perforant pathway stimulation.

(A) Twelve seconds of activity, demonstrating low frequency oscillations (LFOs) at a rate of 1 per second, with a frequency of 13.0 Hz. (B) Spontaneous unilateral EPSP with population spikes recorded from the granule cell layer. (C) Waveform evoked by 7.8 V perforant pathway stimulation. Note the high degree of similarity to panel B. All responses were obtained from the same rat three days post-stimulation. (D) Power Spectrum Density plot showing two minutes of LFOs (red), two minutes of baseline EEG in the same rat (blue), and two minutes of baseline EEG in non-epileptic control (green). Note the greater Power at 1 Hz and from 10–20 Hz, corresponding to the rate and frequency of LFO waveforms (red trace). Calibration bars = 1 s, 10 mV in A; 10 ms, 10 mV in B and C; 10 kHz sampling rate.

Figure 2. Synchronous and asynchronous low frequency oscillations (LFOs) recorded bilaterally from the dentate granule cell layer during the latent period.

(A) A single LFO detected bilaterally. Note the high degree of synchrony within the left hippocampus as well as between hippocampi. (B) Nonsynchronous, bilateral LFOs. All events that exceeded the detection threshold are marked with arrows. Most, but not all, events that occurred in the left hippocampus were detected by both electrodes (L Hipp 1 and 2) which were separated by 2 mm laterally. All traces were obtained from the same freely moving rat twelve days post-stimulation, which was before the first spontaneous seizure. Calibration bars = 2 s, 5 mV; sampling rate 10 kHz.

Figure 3. Low frequency oscillation (LFO) incidence in the dentate gyrus following 8 h perforant pathway stimulation (PPS).

(A) The average number of LFOs detected per day during the latent period in sham perforant pathway transection (PPT) (8 h PPS, no PPT), PPT, and Control (8 h PPS, no PPT, did not develop epilepsy) (n = 4 per group). LFO occurrence was essentially the same in both Sham PPT and PPT groups (p > 0.05). No LFOs were detected in the Control group. (B) Average LFO occurrence per hour on seizure-free days (orange) and during the 60 minutes immediately prior to a spontaneous seizure (green). LFO occurrence usually decreased markedly in the hour preceding a spontaneous seizure (p < 0.001).

Figure 4. Spontaneous hippocampal-onset seizures observed in sham perforant pathway transection (Sham PPT) and PPT rats after 8 h of perforant pathway stimulation.

Traces (A) (Sham PPT) and (B) (PPT) represent 65 seconds of activity recorded from the hippocampal granule cell layer in freely moving rats. Asterisks denote the first overt seizure behavior (forepaw clonus leading to rearing). (C) Mean latency to the first spontaneous seizure. The first seizures manifested after an average of 16.8 + 1.9 d (n = 6) in Sham PPT animals and 15.2 + 1.5 d in the PPT group (n = 5). (D) Mean spontaneous seizure duration. A total of twenty-six seizures were recorded from Sham PPT and PPT animals. Mean seizure length was 77 seconds for Sham PPT and 87 seconds for PPT rats. PPT had no affect on either the length of time between PPS and the first seizure (latent period) or seizure duration (p > 0.05). Calibration bars: 3 s, 4 mV.

Figure 5. Hippocampal neuropathology ca.

2 months after 8 h perforant pathway stimulation in control (A), sham perforant pathway transection (Sham PPT, (B), and PPT (C) groups. NeuN-immunostained transverse sections demonstrating similar neuron loss in both experimental groups. Note the severe loss of CA3 and CA1 neurons in the hippocampus. Arrows in panel C denote the location of the mechanical lesion. (D) Quantification of hippocampal neuron loss. Neuron counts were performed on matching NeuN-immunostained sections. CA3 and CA1 were combined as the border between these regions was unclear in experimental animals. Although substantial neuron loss was seen in the hilus (h) and CA regions in both Sham PPT and PPT groups when compared with control (n = 5 per group, p < 0.001), there was no significant difference between groups (p > 0.05), demonstrating no effect of PPT on neurodegeneration.

Figure 6. Electrophysiological confirmation of perforant pathway transection (PPT) efficacy in vivo.

Representative field potentials recorded from the dentate gyrus, evoked by ipsilateral 0.2 Hz perforant pathway stimulation (PPS) at 20 V in (A): experimental (8 h PPS + PPT) and (B): control (8 h PPS + sham PPT) rats. The small waveform in panel A shows neither a large, positive wave (EPSP) nor population spikes, suggesting that PPT was effective. This is in contrast to the large, complex waveform seen in panel B, which is indicative of effective PPS, i.e. intact entorhinal cortex input to the dentate gyrus. Each panel represents the average of 10 responses.