The use of enzyme-selective microelectrodes for the real-time amperometric detection of neurotransmitter release has generated new insights in the regulation and function of major neurotransmitter and -modulator systems. We previously demonstrated that transient increases in prefrontal cholinergic activity (scale of seconds) mediate the detection of cues in attention-demanding contexts. This research was designed to determine if the transgenic expression of the photo-sensitive cation channel channelrhodopsin-2 (ChR2) is conducive to electrochemical studies measuring neurotransmitter release in the terminal fields of cholinergic projection neurons. We employed a viral vector to selectively express the ChR2 transgene in cholinergic neurons of the basal forebrain (substantia innominata/nucleus basalis of Meynert). Expression was achieved through the infusion of a DIO-ChR2-YFP construct packaged in an AAV vector into mice expressing the CRE recombinase gene under the control of the ChAT promoter. We employed enzyme-coated microelectrodes and fixed potential amperometry in order to measure the release of acetylcholine evoked by photoactivation of cholinergic neurons. Currents recorded via choline oxidase-coated platinum sites were referenced to adjacent non-coated sites. Light pulses were delivered to the cells expressing ChR2 via a laser diode with a wavelength of 446 nm coupled to a fiber-optic cable (200 µm in diameter) that could be raised or lowered via a micromanipulator on a stereotaxic instrument. Individual light pulses (<1 ms in duration, 5-40 mW as measured at the fiber tip) were insufficient to drive detectable cholinergic transients; however short pulses of light delivered in succession (10-60X) at frequencies greater than 10 Hz resulted in measurable cholinergic signals of 0.5 µM or greater. Electrochemical recordings performed in cortical areas contralateral to the hemisphere expressing ChR2 as well as non-projection ipsilateral regions - such as the striatum - did not yield detectable cholinergic signaling. We conclude that the expression of photoactivated ChR2 can be used to selectively activate cholinergic projections to terminal regions and that this release is detectable using enzyme-selective microelectrodes. Future studies will test the hypothesis that the augmentation of cue evoked cholinergic transients improves attentional task-performance and will yield information about the impact of poorly orchestrated or invalid cholinergic transients on cognitive performance.

Optogenetically-evoked cortical cholinergic transients in mice expressing channelrhodopsin-2 (ChR2) in cholinergic neurons.

Paolone G
Investigation
;
2010-01-01

Abstract

The use of enzyme-selective microelectrodes for the real-time amperometric detection of neurotransmitter release has generated new insights in the regulation and function of major neurotransmitter and -modulator systems. We previously demonstrated that transient increases in prefrontal cholinergic activity (scale of seconds) mediate the detection of cues in attention-demanding contexts. This research was designed to determine if the transgenic expression of the photo-sensitive cation channel channelrhodopsin-2 (ChR2) is conducive to electrochemical studies measuring neurotransmitter release in the terminal fields of cholinergic projection neurons. We employed a viral vector to selectively express the ChR2 transgene in cholinergic neurons of the basal forebrain (substantia innominata/nucleus basalis of Meynert). Expression was achieved through the infusion of a DIO-ChR2-YFP construct packaged in an AAV vector into mice expressing the CRE recombinase gene under the control of the ChAT promoter. We employed enzyme-coated microelectrodes and fixed potential amperometry in order to measure the release of acetylcholine evoked by photoactivation of cholinergic neurons. Currents recorded via choline oxidase-coated platinum sites were referenced to adjacent non-coated sites. Light pulses were delivered to the cells expressing ChR2 via a laser diode with a wavelength of 446 nm coupled to a fiber-optic cable (200 µm in diameter) that could be raised or lowered via a micromanipulator on a stereotaxic instrument. Individual light pulses (<1 ms in duration, 5-40 mW as measured at the fiber tip) were insufficient to drive detectable cholinergic transients; however short pulses of light delivered in succession (10-60X) at frequencies greater than 10 Hz resulted in measurable cholinergic signals of 0.5 µM or greater. Electrochemical recordings performed in cortical areas contralateral to the hemisphere expressing ChR2 as well as non-projection ipsilateral regions - such as the striatum - did not yield detectable cholinergic signaling. We conclude that the expression of photoactivated ChR2 can be used to selectively activate cholinergic projections to terminal regions and that this release is detectable using enzyme-selective microelectrodes. Future studies will test the hypothesis that the augmentation of cue evoked cholinergic transients improves attentional task-performance and will yield information about the impact of poorly orchestrated or invalid cholinergic transients on cognitive performance.
2010
Optogenetic
enzyme-selective microelectrodes
Prefrontal cholinergic neurotransmission
Cue-detection
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/988662
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