Paul S. Buckmaster

Objective: To determine whether some cats with epilepsy and temporal lobe epilepsy (TLE)-like seizures have dentate gyrus neuropathologic changes consistent with TLE, including hilar neuron loss and mossy fiber sprouting. Methods: Postmortem hippocampal tissue from 5 cats with a history of seizures and 6 control cats was evaluated. Nissl-stained sections were used to estimate hilar neuron populations within the dentate gyrus by optical fractionator stereology. Timm-stained sections were scored by masked investigators to assess mossy fiber sprouting. Hippocampi from epileptic cats were evaluated bilaterally, and findings were compared with control cats. Results: Mean hilar neuron counts were lower in epileptic cats than controls, although this difference was not statistically significant. Two epileptic cats had hippocampal hilar neuron counts more than 2 SDs below the control mean. Mean Timm scores were higher in epileptic cats than controls, and 2 epileptic cats had mossy fiber sprouting scores more than 2 SDs above the control mean. One epileptic cat had both marked left-sided hilar neuron loss and severe mossy fiber sprouting, greatest in the left hippocampus. Conclusions: The study found that 1 epileptic cat with TLE-like seizures had dentate gyrus neuropathologic changes resembling those described in TLE in humans and other animal models. Clinical Relevance: These findings support naturally occurring feline TLE as a potential clinical entity, and antemortem diagnostics should be investigated to identify these cats for advanced treatment options, such as surgical or laser ablation of the affected hippocampus.

A possible mechanism of temporal lobe epilepsy is insufficient inhibition of hippocampal dentate granule cells. Precipitating injuries that kill interneurons in the dentate gyrus might result in fewer inhibitory synapses with granule cells. To test this hypothesis, previous studies evaluated numbers or densities of interneurons, γ-amino butyric acid (GABA)ergic boutons, and inhibitory synapses in tissue from human patients with temporal lobe epilepsy and rodent models. However, those studies have limitations. Some of those limitations can be addressed by a large animal model. Sea lions ( Zalophus californianus ) can develop temporal lobe epilepsy naturally. Like humans, epileptic sea lions exhibit bilateral or unilateral hippocampal sclerosis (neuron loss) with granule cell vulnerability, but sea lions permit optimal tissue preservation and sampling, and good control subjects. To label interneuron cell bodies and GABAergic synaptic boutons, sea lion hippocampal tissue from both sexes was processed with immunohistochemistry for glutamic acid decarboxylase (GAD) and vesicular GABA transporter. Stereological techniques were used to evaluate the dentate gyrus of the entire hippocampus. Numbers of granule cells, GAD cells, and GABAergic boutons were substantially reduced in shrunken, sclerotic hippocampi. However, numbers of GABAergic boutons and granule cells were correlated. These findings indicate that, despite losses, numbers of GABAergic boutons scale with numbers of surviving granule cells.

Objective: To evaluate neuropathological changes in dogs with idiopathic epilepsy and history of focal seizures using previously described criteria for temporal lobe epilepsy (TLE) in humans and other animals. Methods: This was a prospective descriptive study. Dogs of any age, sex, or breed with a history of idiopathic epilepsy and focal seizures were included. Immediately following euthanasia, dogs were perfused, and tissue was collected. Brains were sectioned, mounted, and stained. Nissl-stained slides were used for stereology, and Timm stain was used to evaluate for evidence of mossy fiber sprouting. Results: 7 dogs were included in the final analysis. For stereological cell counts evaluating hilar neuron loss, no case was significantly lower in number of neurons. Additional analysis compared right and left to look for asymmetry, and none were significant ( P = .259). Using Timm stain to evaluate mossy fiber sprouting, no cases showed extensive sprouting, and no asymmetry was detected between left and right hippocampi ( P = .054). Conclusions: In our cohort, while there were changes associated with seizures detected within the hippocampi, none were severe or consistent enough to qualify as TLE using the human criteria. Clinical Relevance: Our study focused on dogs with focal seizures as their main seizure presentation as that is the most common semiology in people with TLE. However, a future study could consider evaluating the neuropathology of dogs with asymmetric hippocampi detected on MRI. If TLE can be shown in dogs, then advanced treatment options, such as surgery or laser ablation, could be considered in refractory patients.

Temporal lobe epilepsy is common, but mechanisms of seizure initiation are unclear. We evaluated seizure initiation in female and male rats that had been systemically treated with pilocarpine, a widely used model of temporal lobe epilepsy. Local field potential (LFP) recordings from many brain regions revealed variable sites of earliest recorded seizure activity, but mostly the ventral hippocampal formation. To test whether inactivation of the ventral hippocampal formation would reduce seizures, mini-osmotic pumps were used to continually and focally deliver TTX. High doses of TTX infused unilaterally into the ventral hippocampal formation blocked seizures reversibly but also reduced LFP amplitudes in remote brain regions, indicating distant effects. A lower dose did not reduce LFP amplitudes in remote brain regions but did not reduce seizures when infused unilaterally. Instead, seizures tended to initiate in the contralateral ventral hippocampal formation. Bilateral infusion of the lower dose into the ventral hippocampal formation reduced seizure frequency 85%. Similar bilateral treatment in the amygdala was not effective. Bilateral infusion of the dorsal hippocampus reduced seizure frequency, but only 17%. Together, these findings reveal that the ventral hippocampal formation is a primary bilaterally independent epileptogenic zone, and the dorsal hippocampus is a secondary epileptogenic zone in pilocarpine-treated rats. This is consistent with many human patients, and the results further validate the LFP method for identifying seizure onset zones. Finally, the findings are more consistent with a focal mechanism of ictogenesis rather than one involving a network of interdependent nodes. SIGNIFICANCE STATEMENT To better understand how seizures start, investigators need to know where seizures start in the animal models they study. In the widely used pilocarpine-treated rat model of temporal lobe epilepsy, earliest seizure activity was most frequently recorded in the ventral hippocampal formation. Confirming the primary role of the ventral hippocampal formation, seizure frequency was reduced most effectively when it was inactivated focally, bilaterally, and continually with infused TTX. These findings suggest that the ventral hippocampal formation is the primary site of seizure initiation in this animal model of temporal lobe epilepsy, consistent with findings in many human patients.

Temporal lobe epilepsy remains a common disorder with no cure and inadequate treatments, potentially because of an incomplete understanding of how seizures start. CA1 pyramidal cells and many inhibitory interneurons increase their firing rate in the seconds-minutes before a spontaneous seizure in epileptic rats. However, some interneurons fail to do so, including those identified as putative interneurons with somata in oriens and axons targeting lacunosum-moleculare (OLM cells). Somatostatin-containing cells, including OLM cells, are the primary target of inhibitory vasoactive intestinal polypeptide and calretinin-expressing (VIP/CR) bipolar interneuron-selective interneurons, type 3 (ISI-3). The objective of this study was to test the hypothesis that in epilepsy inhibition of OLM cells by ISI-3 is abnormally increased, potentially explaining the failure of OLM recruitment when needed most during the ramp up of activity preceding a seizure. Stereological quantification of VIP/CR cells in a model of temporal lobe epilepsy demonstrated that they survive in epileptic mice, despite a reduction in their somatostatin-expressing (Som) cell targets. Paired recordings of unitary IPSCs (uIPSCs) from ISI-3 to OLM cells did not show increased connection probability or increased connection strength, and failure rate was unchanged. When miniature postsynaptic currents in ISI-3 were compared, only mIPSC frequency was increased in epileptic hippocampi. Nevertheless, spontaneous and miniature postsynaptic potentials were unchanged in OLM cells of epileptic mice. These results are not consistent with the hypothesis of hyperinhibition from VIP/CR bipolar cells impeding recruitment of OLM cells in advance of a seizure.

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.

One in 26 people develop epilepsy and in these temporal lobe epilepsy (TLE) is common. Many patients display a pattern of neuron loss called hippocampal sclerosis. Seizures usually start in the hippocampus but underlying mechanisms remain unclear. One possibility is insufficient inhibition of dentate granule cells. Normally parvalbumin‐immunoreactive (PV) interneurons strongly inhibit granule cells. Humans with TLE display loss of PV interneurons in the dentate gyrus but questions persist. To address this, we evaluated PV interneuron and bouton numbers in California sea lions (Zalophus californianus) that naturally develop TLE after exposure to domoic acid, a neurotoxin that enters the marine food chain during harmful algal blooms. Sclerotic hippocampi were identified by the loss of Nissl‐stained hilar neurons. Stereological methods were used to estimate the number of granule cells and PV interneurons per dentate gyrus. Sclerotic hippocampi contained fewer granule cells, fewer PV interneurons, and fewer PV synaptic boutons, and the ratio of granule cells to PV interneurons was higher than in controls. To test whether fewer boutons was attributable to loss versus reduced immunoreactivity, expression of synaptotagmin‐2 (syt2) was evaluated. Syt2 is also expressed in boutons of PV interneurons. Sclerotic hippocampi displayed proportional losses of syt2‐immunoreactive boutons, PV boutons, and granule cells. There was no significant difference in the average numbers of PV‐ or syt2‐positive boutons per granule cell between control and sclerotic hippocampi. These findings do not address functionality of surviving synapses but suggest reduced granule cell inhibition in TLE is not attributable to anatomical loss of PV boutons.

Epilepsy occurs in one of 26 people. Temporal lobe epilepsy is common and can be difficult to treat effectively. It can develop after brain injuries that damage the hippocampus. Multiple pathophysiological mechanisms involving the hippocampal dentate gyrus have been proposed. This study evaluated a mouse model of temporal lobe epilepsy to test which pathological changes in the dentate gyrus correlate with seizure frequency and help prioritize potential mechanisms for further study. FVB mice (n = 127) that had experienced status epilepticus after systemic treatment with pilocarpine 31–61 days earlier were video‐monitored for spontaneous, convulsive seizures 9 hr/day every day for 24–36 days. Over 4,060 seizures were observed. Seizure frequency ranged from an average of one every 3.6 days to one every 2.1 hr. Hippocampal sections were processed for Nissl stain, Prox1‐immunocytochemistry, GluR2‐immunocytochemistry, Timm stain, glial fibrillary acidic protein‐immunocytochemistry, glutamic acid decarboxylase in situ hybridization, and parvalbumin‐immunocytochemistry. Stereological methods were used to measure hilar ectopic granule cells, mossy cells, mossy fiber sprouting, astrogliosis, and GABAergic interneurons. Seizure frequency was not significantly correlated with the generation of hilar ectopic granule cells, the number of mossy cells, the extent of mossy fiber sprouting, the extent of astrogliosis, or the number of GABAergic interneurons in the molecular layer or hilus. Seizure frequency significantly correlated with the loss of GABAergic interneurons in or adjacent to the granule cell layer, but not with the loss of parvalbumin‐positive interneurons. These findings prioritize the loss of granule cell layer interneurons for further testing as a potential cause of temporal lobe epilepsy.