Selective loss of dentate hilar interneurons contributes to reduced synaptic inhibition of granule cells in an electrical stimulation-based animal model of temporal lobe epilepsy

Abstract

Neuropeptide-containing hippocampal interneurons and dentate granule cell inhibition were investigated at different periods following electrical stimulation-induced, self-sustaining status epilepticus (SE) in rats. Immunohistochemistry for somatostatin (SOM), neuropeptide Y (NPY), parvalbumin (PV), cholecystokinin (CCK), and Fluoro-Jade B was performed on sections from hippocampus contralateral to the stimulated side and studied by confocal laser scanning microscopy. Compared to paired age-matched control animals, there were fewer SOM and NPY-immunoreactive (IR) interneurons in the hilus of the dentate gyrus in animals with epilepsy (40-60 days after SE), and 1, 3, and 7 days following SE. In the hilus of animals that had recently undergone SE, some SOM-IR and NPY-IR interneurons also stained for Fluoro-Jade B. Furthermore, there was electron microscopic evidence of the degeneration of SOM-IR interneurons following SE. In contrast, the number of CCK and PV-IR basket cells in epileptic animals was similar to that in controls, although it was transiently diminished following SE; there was no evidence of degeneration of CCK or PV-IR interneurons. Patch-clamp recordings revealed a diminished frequency of inhibitory postsynaptic currents in dentate granule cells (DGCs) recorded from epileptic animals and animals that had recently undergone SE compared with controls. These results confirm the selective vulnerability of a particular subset of dentate hilar interneurons after prolonged SE. This loss may contribute to the reduced GABAergic synaptic inhibition of granule cells in epileptic animals.

Fig. 1

There were fewer somatostatin (SOM)-immunoreactive (IR) cells in the dentate hilus in ventral hippocampal sections obtained from animals 1 day (B), 3 days (C), 7 days (D) post-status epilepticus (SE) and in epileptic animals (TLE, E) compared to age-matched controls (A). F: Pairwise comparisons of number of SOM-IR cells in hilus of hippocampi from control and SE-treated animals (P < 0.05). G: The number of SOM-IR cells in the hilus (expressed as percent controls) in animals 1 day, 3 days, and 7 days following SE and in epileptic animals. D1, 1 day after SE; D3, 3 days after SE; D7, 7 days after SE; TLE, temporal lobe epilepsy. Scale bar = 50 μm.

Fig. 2

Hippocampal sections obtained from animals 24 hours following SE, stained for Fluoro-Jade B and SOM, or neuropeptide Y (NPY), or parvalbumin (PV), or cholecystokinin (CCK). Some SOM and NPY-IR hilar interneurons also stained for Fluoro-Jade B but none of the CCK or PV-IR cells stained for Fluoro-Jade B. A,D,G,J: Fluoro-Jade B stained cells in hilus. B,E,H,K: SOM, NPY, PV, and CCK-IR cells in the hilus, respectively. C,F,I,L: Digitally merged images. Some SOM or NPY-IR interneurons were stained with Fluoro-Jade B also (A–F; arrowhead shows the double-labeled cells). None of the PV and CCK-IR interneurons stained for Fluoro-Jade B. Scale bars = 20 μm in A–F; 50 μm in G–L.

Fig. 3

Electron micrographs of preembedding silver-enhanced staining of SOM-IR interneurons from hippocampus of a control animal (A) and an animal 7 days post-SE (B). In B, note perinuclear vacuoles in SOM-IR cell, which is indicative of early degenerative change. Vacuole in boxed area of B was magnified in D (arrowhead). Corresponding perinuclear area was boxed in A, magnified in C. Scale bar = 1 μm.

Fig. 4

Electron micrographs demonstrating a normal SOM-IR dendrite from a control (A) and a degenerating dendrite from an animal 7 days post-SE (B). Vacuoles and condensation of mitochondria in SOM-IR dendrites were found in B. Boxed areas in A,B are magnified in C,D, respectively. Scale bar = 0.5 μm.

Fig. 5

There were fewer NPY-IR cells in the dentate hilus in ventral hippocampal sections 3 days (C), 7 days (D) post-SE, and in TLE (E) animals compared to age-matched controls (A). F: Pairwise comparisons of number of NPY-IR cells in hilus of hippocampi from control and SE-treated animals (P < 0.05). G: The number of NPY-IR cells in the hilus, expressed as percent controls in animals 1, 3, and 7 days post-SE and in epileptic animals. Scale bar = 50 μm.

Fig. 6

Compared to age-matched controls, the number PV-IR cells in the dentate hilus in ventral hippocampal sections 1 day (B), 3 days (C), 7 days (D) post-SE was diminished but was unchanged in epileptic animals (E). F: Pairwise comparisons of number of PV-IR cells in hilus of hippocampi from control and SE-treated animals (D1, D3, D7, P < 0.05; TLE, P > 0.05). G: The number of PV-IR cells in the hilus (expressed as percent controls) at 1, 3, 7 days post-SE was diminished (P < 0.05) and was not changed in TLE animals (E). Scale bar = 50 μm.

Fig. 7

Compared to age-matched controls, the number CCK-IR cells in the dentate hilus in ventral hippocampal sections 3 days (C), 7 days (D) post-SE was diminished but was unchanged in 1 day post-SE (B) and epileptic animals (E). F: Pairwise comparisons of number of CCK-IR cells in hilus of hippocampi from control and SE-treated animals (D3 and D7, P < 0.05; D1 and TLE, P > 0.05). G: The number of CCK-IR cells in the hilus (expressed as percent controls) at 1, 3, 7 days post-SE and in epileptic animals. Scale bar = 50 μm.

Fig. 8

Frequency of sIPSCs recorded from DGCs in hippocampal slices from animals 1, 3, and 7 days following SE and from epileptic animals. A: Recording from control DGC. a: Fragments from the same recording sessions as in A but with a higher temporal resolution. Horizontal scale bars show time; vertical bars show current amplitude in pA. B,b: Recording from a DGC from a rat 7 days after SE. C,c: Recording from a DGC from a rat with TLE. Note decreased frequency of sIPSCs in TLE. D: Mean frequency of sIPSCs decreased progressively after SE and was lowest 7 days post-SE. There was a slight but not significant increase of frequency in TLE compared to day 7. E: Frequency of mIPSCs decreased following SE and remained decreased in TLE. Note the similar pattern of frequency decrease of mIPSCs and sIPSCs, suggesting reduction in number of release sites. Each point represents mean Hz ± SEM. F: Correlation between decrease in number of SOM-IR cells and reduction of frequency of sIPSCs after SE. Abscissa shows number of SOM-IR cells is shown as percentage of surviving cells where 100% is taken as a control. Ordinate shows frequency of sIPSCs in Hz. Each point represents time after SE.

Fig. 9

Frequency distribution histograms of 10 –90% rise times of mIPSCs recorded from DGC from a control (A), 7 days post-SE (B) and in TLE (C) animals. Approximately 7,500 mIPSCs from 5– 6 cells were pooled in control and TLE groups. Approximately 1,300 events were pooled from four DGCs in animals 7 days post-SE. Rise times were binned to 0.1-ms bin size and fit to multiple Gaussian distributions. Curves show fast (light gray), intermediate (black), and slow (dark gray) rise time distributions. Proportions of the events in three populations are displayed in corresponding pie charts where light gray, black, and dark gray segments correspond to fast, intermediate, and slow rise times. Note that slow rise time events were markedly diminished 7 days post-SE. There was a reduction of proportion of slow rise time events and an increase of proportion of intermediate events in TLE compared to control.