Abstracts

Rationale:
Tetramethylenedisulfotetramine (TETS), a potent rodenticide and GABAA inhibitor is a chemical terrorist threat agent that can produce severe seizures, progressive neurodegeneration, and neurocognitive deficits in humans and animals for which sufficient treatment options are lacking. We hypothesized that Ca2+dysregulation, a critical element in the toxicity of these agents, is reversible by the non-psychotropic cannabinoid, cannabidiol (CBD). Evaluation of neuronal cell survival and cell death signaling pathways regulated by Ca2+ in response to TETS has not been explored nor as to how it interacts with CBD singly or in combination with other anti-epileptic drugs (AEDs). Therefore, an in vitro approach comparing hippocampal vs. cortical cultures was taken.
Method:
At 14 DIV, dissociated hippocampal and sister cortical cultures were exposed to TETS (10 µM) ± CBD (15 µM) ± MDZ (10 µM) for 24 h. Expression of a Ca2+ binding protein calmodulin 2 (Calm2), a Ca2+ specific controller of several signaling proteins and two high voltage-gated Ca2+ channels (VGCC), P/Q and Cav1.2 (L type) to which they bind with specific antibodies were immunocytochemically investigated. [Ca2+]i was measured with a Flexstation 3 at 25oC at varied doses of CBD and MDZ following acute TETS application.
Results:
After a 24 h exposure, TETS reduced Calm2 immunostaining in darkly and lightly stained hippocampal neurons; many cells were shrunken. When TETS was co-treated with CBD, hippocampal Calm2 and P/Q expression were elevated and darkly-stained neurons were increased; morphology was preserved. In contrast, midazolam (MDZ, 10 µM), a GABAA positive modulator, reduced Calm2 expression and further reduced P/Q positive cell counts at the concentration tested. Cav1.2 immunostaining increased after TETS in hippocampal neurons with compromised morphology, which was reversed with CBD or MDZ. Cortical neuronal cultures grown under the same conditions were unaffected by any of the treatments.  Application of CBD (50 μM) + MDZ (5μM) after acute TETS application produced greater suppression of [Ca2+]i signals than either agent alone. At 100 μM CBD, suppression on [Ca2+]I was maximal in the absence of MDZ.
Conclusion:
Results suggest a novel mechanism of CBD neuroprotection against severe seizures by suppression of persistent elevations in [Ca2+]i and upregulation of critical Ca2+ binding proteins. Hippocampal neurons are more sensitive to TETS toxicity than cortical neurons so that lower non-saturating doses of MDZ would be required to avoid adversely affecting hippocampal Ca2+ binding proteins. High doses of CBD induced maximal suppression of [Ca2+]i signals suggesting CBD alone may improve the standard of care for chemical threats.
Funding:
:None