Ictal onset sites and γ-aminobutyric acidergic neuron loss in epileptic pilocarpine-treated rats

Abstract

Objective: The present study tested whether ictal onset sites are regions of more severe interneuron loss in epileptic pilocarpine-treated rats, a model of human temporal lobe epilepsy.

Methods: Local field potential recordings were evaluated to identify ictal onset sites. Electrode sites were visualized in Nissl-stained sections. Adjacent sections were processed with proximity ligation in situ hybridization for glutamic acid decarboxylase 2 (Gad2). Gad2 neuron profile numbers at ictal onset sites were compared to contralateral regions. Other sections were processed with immunocytochemistry for reelin or nitric oxide synthase (NOS), which labeled major subtypes of granule cell layer-associated interneurons. Stereology was used to estimate numbers of reelin and NOS granule cell layer-associated interneurons per hippocampus.

Results: Ictal onset sites varied between and within rats but were mostly in the ventral hippocampus and were frequently bilateral. There was no conclusive evidence of more severe Gad2 neuron profile loss at sites of earliest seizure activity compared to contralateral regions. Numbers of granule cell layer-associated NOS neurons were reduced in the ventral hippocampus.

Significance: In epileptic pilocarpine-treated rats, ictal onset sites were mostly in the ventral hippocampus, where there was loss of granule cell layer-associated NOS interneurons. These findings suggest the hypothesis that loss of granule cell layer-associated NOS interneurons in the ventral hippocampus is a mechanism of temporal lobe epilepsy.

Keywords: epilepsy; focus; hippocampus; interneuron; seizure.

Figure 1

Earliest recorded seizure activity at multiple sites in an epileptic pilocarpine-treated rat. A Seizure onset indicated by red dashed line. B Seizure 1 is an expanded view that reveals earliest recorded seizure activity in the left ventral subiculum. The high-amplitude background activity in the right ventral hippocampus should not be confused with the seizure onset. Seizures 2 and 3 show onsets of two other seizures recorded on another day. The earliest recorded seizure activity was in the right ventral subiculum of seizure 2 and the left dorsal hippocampus of seizure 3.

Figure 2

Earliest recorded seizure activity in the ventral hippocampus. A Seizure onset indicated by red dashed line. B Expanded view reveals earliest seizure activity in the right ventral hippocampus. C Nissl-stained section reveals long (arrow) and short parts of a bipolar electrode track (double arrow). The short electrode recorded the earliest seizure activity 118 times, which was 96% of all seizures recorded earliest in the right ventral hippocampus and 63% of all seizures recorded in this rat. m = molecular layer, g = granule cell layer, h = hilus, S = subiculum.

Figure 3

Glutamic acid decarboxylase 2 (Gad2) neurons at ictal onset sites. A Nissl-stained section with a box denoting a 250 μm radius centered on the electrode tip with the highest z-score for recording earliest seizure activity. m = molecular layer, g = granule cell layer, h = hilus, S = subiculum. B Gad2 neurons in the dentate gyrus of a control rat. DAPI in blue. C Gad2 neurons at the ictal onset site centered on the electrode tip as demarcated in A but in a nearby section. D Gad2 in situ at the contralateral site. E Quantification of Gad2 neuron profiles at the ictal onset site and contralaterally for each rat. Averages indicated by horizontal black lines. Error bars are s.e.m. Magenta lines indicate rats in which the contralateral site was not a site of significantly earliest seizure activity.

Figure 4

Cells that express reelin or nitric oxide synthase account for most granule cell layer-associated interneurons. A-G Adjacent hippocampal sections from a control rat processed for different interneuron markers. m = molecular layer, g = granule cell layer, h = hilus. H Number of interneurons in or adjacent to the granule cell layer per hippocampus. Circles represent individuals, lines indicate averages.

Figure 5

Granule layer-associated reelin (RLN)- and nitric oxide synthase (NOS)-immunoreactive interneurons. Ventral hippocampus in a control (A,B) and epileptic rat (C,D). m = molecular layer, g = granule cell layer, h = hilus. E Method to analyze coronal sections. Nissl stained sections from a control rat show the granule cell layer analyzed at different levels of the septotemporal axis of the hippocampus: red outline = septal (dorsal) dentate gyrus; green outline = temporal (ventral) dentate gyrus. E2,6 are the same section. E3,5 are the same section. E4 was the most posterior section that included granule cell layer. Results from the dorsal and ventral parts of the granule cell layer were plotted as section −1 and 1, respectively, in panels F,G. Granule cell layer-associated RLN- (F) and NOS-positive neuron profiles/section (G). Number of granule cell layer associated RLN- (H) and NOS-positive neurons (I) in the entire hippocampus (total), dorsal hippocampus, and ventral hippocampus. *p < 0.01, **p < 0.001, t test. For NOS, there also is a plot of the number in ventral hippocampi of epileptic rats without versus with a significant ictal onset site.