Ethanol Inhibition of Up-States in Prefrontal Cortical Neurons Expressing the Genetically Encoded Calcium Indicator GCaMP3

摘要

Background: The prefrontal cortex (PFC) is critically involved in working memory, cognition, and decision making; processes significantly affected by ethanol (EtOH). During quiet restfulness or sleep, PFC neurons show synaptically evoked oscillations in membrane potential between hyperpolarized down-states and depolarized up-states. Previous studies from this laboratory used whole-cell electrophysiology and demonstrated that in individual neurons, EtOH inhibited PFC up-states at concentrations associated with behavioral impairment. Although those studies monitored activity in 1 or 2 neurons at a time, it is likely that in vivo, larger networks of neurons participate in the complex functions of the PFC. In the present study, we used imaging and a genetically encoded calcium sensor to examine the effects of EtOH on the activity of multiple neurons simultaneously during up-states. Methods: Slice cultures of mouse PFC were infected with an AAV virus encoding the calcium indicator GCaMP3 whose expression was driven by the neuron-specific synapsin promoter. After 2 to 3weeks in culture, a fast CCD-camera imaging system was used to capture changes in GCaMP3 fluorescence before, during, and after exposure to EtOH. Results: PFC neurons displayed robust and reproducible changes in GCaMP3 fluorescence during evoked and spontaneous up-states. Simultaneous whole-cell patch-clamp recording and GCaMP3 imaging verified that neurons transitioned into and out of up-states together. Acute application of EtOH reliably depressed up-state calcium signals with lower doses having a greater effect on up-state duration than amplitude. These effects of EtOH on up-state parameters were reversed during washout. Conclusions: The results of the present study indicate that EtOH has profound effects on up-state activity in prefrontal neurons and suggest that this action may underlie some of the cognitive impairment associated with acute alcohol intoxication.