Visualizing protein kinase A activity in head-fixed behaving mice using in vivo two-photon fluorescence lifetime imaging microscopy

Bart C. Jongbloets, Lei Ma, Tianyi Mao, Haining Zhong

Research output: Contribution to journalArticle

Abstract

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKARα, is used, which is based on Förster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKARα is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

Original languageEnglish (US)
Article numbere59526
JournalJournal of Visualized Experiments
Volume2019
Issue number148
DOIs
StatePublished - Jun 1 2019

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Optical Imaging
Cyclic AMP-Dependent Protein Kinases
Photons
Microscopy
Microscopic examination
Fluorescence
Energy Transfer
Head
Proteins
Imaging techniques
Energy transfer
Brain
Neurotransmitter Agents
Norepinephrine
Exercise equipment
Dependovirus
Electroporation
Sensors
Motor Cortex
Locomotion

Keywords

  • A-kinase activity reporter (AKAR)
  • CAMP-dependent protein kinase/protein kinase A (PKA)
  • Craniotomy
  • Förster resonance energy transfer (FRET)
  • In vivo two-photon fluorescence lifetime imaging microscopy (2pFLIM)
  • Issue 148
  • Locomotion
  • Neuromodulation
  • Neuroscience
  • TAKARα

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Cite this

Visualizing protein kinase A activity in head-fixed behaving mice using in vivo two-photon fluorescence lifetime imaging microscopy. / Jongbloets, Bart C.; Ma, Lei; Mao, Tianyi; Zhong, Haining.

In: Journal of Visualized Experiments, Vol. 2019, No. 148, e59526, 01.06.2019.

Research output: Contribution to journalArticle

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abstract = "Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKARα, is used, which is based on F{\"o}rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKARα is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.",
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