Optogenetic Determination of Dynamic and Cell-Type-Specific Inhibitory Reversal Potentials.

The reversal potential refers to the membrane potential at which the net current flow through a channel reverses direction. The reversal potential is determined by transmembrane ion gradients and, in turn, determines how the channel's activity will affect the membrane potential. Traditional investigation into the reversal potential of inhibitory ligand-gated ion channels (EInh) has relied upon the activation of endogenous receptors, such as the GABA-A receptor (GABAAR). There are, however, challenges associated with activating endogenous receptors, including agonist delivery, isolating channel responses, and the effects of receptor saturation and desensitization. Here, we demonstrate the utility of using a light-gated anion channel, stGtACR2, to probe EInh in the rodent brain. Using mice of both sexes, we demonstrate that the properties of this optically activated channel make it a suitable proxy for studying GABAAR receptor-mediated inhibition. We validate this agonist-independent optogenetic strategy in vitro and in vivo and further show how it can accurately capture differences in EInh dynamics following manipulations of endogenous ion fluxes. This allows us to explore distinct resting EInh differences across genetically defined neuronal subpopulations. Using this approach to challenge ion homeostasis mechanisms in neurons, we uncover cell-specific EInh dynamics that are supported by the differential expression of endogenous ion handling mechanisms. Our findings therefore establish an effective optical strategy for revealing novel aspects of inhibitory reversal potentials and thereby expand the repertoire of optogenetics.

Dysregulation of astrocytic mitochondrial function following exposure to a dopamine metabolite: Implications for Parkinson's disease.

The monoamine oxidase metabolite of dopamine, 3,4-dihydroxyphenylacetaldehyde (DOPAL), is hypothesized to induce neurodegeneration in Parkinson's disease (PD). However, DOPAL's effect on astrocyte function is less well known. Furthermore, the conflicting protective and pathological roles of resting and reactive astrocytes in Parkinson's disease have led to astrocytes being characterized as a double-edged sword in this disease. Using the Neu7 rat astrocyte cell line as a model of astrocyte behaviour, we aimed to evaluate the effect of DOPAL on astrocyte viability, reactivity and mitochondrial function. Astrocytic production of hydrogen peroxide and nitrite was indicative of reactivity. Mitochondrial function was assessed using extracellular flux analysis with the Seahorse extracellular flux analysis system and mitochondria membrane potential dye. We found that DOPAL significantly reduces Neu7 viability, induces apoptosis, decreases mitochondrial performance and increases oxidative and nitrative stress in a concentration-dependent manner. This is the first in vitro study showing that DOPAL is directly toxic to astrocytes. We predict that the loss of astrocyte viability and the gain of neurotoxic effects, like the increase in oxidative stress, will have detrimental consequences to neuronal viability. This research supports the hypothesis that DOPAL is a contributing factor to PD progression and provides a basis for future research to elucidate the mechanism of DOPAL-induced astrocyte toxicity in PD.