Abstract

Domoic acid (DA), an excitatory neurotoxic glutamate receptor agonist, is a common cause of disease in free-ranging California sea lions (Zalophus californianus), affecting up to hundreds of animals per year.^1,2 Sea lions that survive acute toxicosis can sustain brain damage leading to temporal lobe epilepsy, which is characterized by behavioral and cognitive changes, hippocampal atrophy, and periodic seizures, clinically described as chronic DA toxicosis.^2-4 Sea lions suffering from temporal lobe epilepsy do not thrive in the wild and are typically euthanized after stranding, while affected individuals in managed care offer complex challenges and may require chronic medication.^4-6 While studies in other mammals have highlighted the importance of hippocampal damage in temporal lobe epilepsy, the precise pathophysiologic mechanism of damage that leads to the brain becoming epileptic is unknown, leading to the proposal of multiple hypothesizes, such as a decrease in inhibitory regulation of excitatory cells in the dentate gyrus region of the hippocampus.^7,8 If the underlying pathophysiology of epileptogenesis can be identified, then novel treatments to prevent or reverse epileptogenesis can be developed. This study aims to test whether there is a loss of inhibitory structures, GABAergic synaptic boutons, in the dentate gyrus of chronic DA sea lions.

Sea lions that presented to The Marine Mammal Center (Sausalito, CA, USA) between 2010–2015 and were non-releasable due to incurable epilepsy (n=31) or terminal non-neurologic disease as controls (n=13) were perfused with 4% formaldehyde immediately following euthanasia. The brains were removed and sectioned for analysis as previously described.⁹ The current study used additional sections of the same brains to quantify GABAergic synaptic boutons. Immunohistochemical staining of vesicular GABA transporter and glutamic acid decarboxylase was used to label GABAergic boutons in the dentate gyrus. Stereology was used to estimate the total number of GABAergic synaptic boutons in each dentate gyrus. Preliminary results showed control hippocampi (n=6) had an average of 530±52 million GABAergic boutons compared to epileptic hippocampi (n=7), averaging only 314±32 million, a 41% decrease (p=0.004, t-test). These findings show a loss of inhibitory synaptic boutons in epileptic hippocampi; however, they do not take into account concurrent losses of target cells. Numbers of excitatory granule cells per dentate gyrus estimated in adjacent Nissl-stained sections using stereology. Control hippocampi had an average of 250±14 million granule cells compared to epileptic hippocampi averaging only 542±212 thousand, a 78% decrease (p<0.001, t-test).

When these data were combined, the average number of GABAergic synaptic boutons per granule cell in the control hippocampi was 216±28, but epileptic hippocampi had an average of 1095±319, or 5.1 times more compared with controls (p=0.005, Wilcoxon rank sum test).

Contrary to the hypothesis, these preliminary results reveal more, not fewer, inhibitory synaptic boutons per excitatory granule cell in epileptic hippocampi. This suggests that a mechanism other than structural loss of inhibitory GABAergic boutons is the primary cause of epileptogenesis in California sea lions with DA-induced temporal lobe epilepsy.

Acknowledgments

The authors thank the staff and volunteers of The Marine Mammal Center for their dedicated animal rescue and care. Funding support from the National Institute of Health grants NS107290, T35OD010989, T32OD11121, and National Science Foundation grant ES021960.

*Presenting author
+Student presenter

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