Abstracts

Rationale: Focal temporal lobe seizures that do not generalize can also impair consciousness. Previous human and rodent intracranial recordings demonstrated low-frequency oscillations in the neocortex during high-frequency, poly-spike seizure activity in the temporal lobe. This cortical slow wave activity is also detected in other impaired consciousness states such as coma, encephalopathy or deep sleep. However, why and how limbic seizures can cause loss of consciousness is unknown. According to the network inhibition hypothesis, temporal lobe seizures may inhibit subcortical arousal systems and thereby cause a transition to cortical slow wave activity. The pedunculopontine tegmental nucleus (PPT) is one of the subcortical arousal-related structures involved in maintaining arousal and REM sleep. Therefore, we hypothesize that the PPT may play a role in mediating the remote effect of the temporal lobe on the cortex during complex partial seizures. Methods: We use optogenetic stimulation of cholinergic PPT neurons in a rat model of complex partial limbic seizures, aiming to reverse slow wave activity in the neocortex. To selectively express channelrhodopsin-2 (ChR2, a light sensitive, nonspecific cation channel) in PPT cholinergic neurons, we stereotaxically injected a Cre-dependent double-floxed ChR2-eYFP virus (rAAV5/ EF1 -DIO-ChR2-eYFP, H134R) unilaterally into the PPT of ChAT-Cre rats that specifically express Cre-recombinase in cholinergic neurons. We then used immunohistochemistry to analyze the colocalization of choline acetyltransferase (ChAT) and eYFP (virus), and used this anatomical approach to optimize injection parameters. We used cell counting to quantify the specificity and the robustness of ChR2 expression in PPT cholinergic neurons. Results: The PPT contains cholinergic (ChAT positive) and non-cholinergic neurons. Following virus injections into the PPT, we found that a large fraction of ChAT positive neurons were stained by eYFP, indicating a high ChR2 expression level in cholinergic neurons. Importantly, nearly all eYFP neurons were stained by ChAT, demonstrating a high expression specificity of ChR2 in the targeted neurons. Based on this histological analysis, stimulating the PPT using laser light would selectively activate cholinergic neurons in this brain region, allowing mechanistic insights into their function during limbic seizures.Conclusions: We found highly specific expression of ChR2 in the targeted cholinergic neurons of the PPT, suggesting that optogenetic stimulation of these neurons will be a promising technique for activating the forebrain during limbic seizures. This strategy will hopefully provide new mechanistic insights into epileptic unconsciousness, and pave the way for novel treatments to restore normal cortical function and prevent loss of consciousness during complex partial seizures.