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

Authors

  • John J. Woodward,

    1. From the Department of Neurosciences and Center for Drug and Alcohol Programs (JJW, MP), Medical University of South Carolina, Charleston, South Carolina.
    Search for more papers by this author
  • Matthew Pava

    1. From the Department of Neurosciences and Center for Drug and Alcohol Programs (JJW, MP), Medical University of South Carolina, Charleston, South Carolina.
    Search for more papers by this author

Reprint requests: John J. Woodward, Departmant of Neurosciences, MUSC IOP 4 North, MSC 861, Charleston, SC 29425; Tel.: 843-792-5225; Fax: 843-792-7353; E-mail:woodward@musc.edu

Abstract

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 3 weeks 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.

Ancillary