A Highly Sensitive A-Kinase Activity Reporter for Imaging Neuromodulatory Events in Awake Mice

Ma, Lei; Jongbloets, Bart C.; Xiong, Wei; Melander, Joshua B.; Qin, Maozhen; Lameyer, Tess J.; Harrison, Madeleine F.; Zemelman, Boris V.; Mao, Tianyi; Zhong, Haining

doi:10.1016/j.neuron.2021.06.006

Cited by 6 publications

(12 citation statements)

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“…4). Given that multiple neuromodulators can be released in the motor cortex 37 , different downstream signaling processes are expected to be induced in cortex neurons, which might partially explain the discrepancy between cAMP signal and calcium activity in our results (Fig. 4f).…”

Section: Discussionmentioning

confidence: 74%

“…To demonstrate the utility of G-Flamp1 sensor to detect physiologically relevant cAMP dynamics in living animals, we performed head-fixed two-photon imaging in the motor cortex (M1) of awake mice during forced locomotion (Fig. 4a), which was reported to be associated with increased neuromodulator and PKA activities 37 . We co-expressed G-Flamp1 (or G-Flamp1-mut) and the red calcium sensor jRGECO1a in the neurons of motor cortex and imaged the layer 2/3 region (Fig.…”

Section: In Vivo Two-photon Imaging Of Camp Dynamics In Mouse Cortexmentioning

confidence: 99%

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A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging

Wang

Peng

et al. 2022

Preprint

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cAMP is a key second messenger that regulates diverse cellular functions including neural plasticity. However, the spatiotemporal dynamics of intracellular cAMP in intact organisms are largely unknown due to low sensitivity and/or brightness of current genetically encoded fluorescent cAMP indicators. Here, we report the development of the new circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1, which exhibits a large fluorescence increase (a maximum ΔF/F0 of 1100% in HEK293T cells), relatively high brightness, appropriate affinity (a Kd of 2.17 μM) and fast response kinetics (an association and dissociation half-time of 0.20 s and 0.087 s, respectively). Furthermore, the crystal structure of the cAMP-bound G-Flamp1 reveals one linker connecting the cAMP-binding domain to cpGFP adopts a distorted β-strand conformation that may serve as a fluorescence modulation switch. We demonstrate that G-Flamp1 enables sensitive monitoring of endogenous cAMP signals in brain regions that are implicated in learning and motor control in living organisms such as fruit flies and mice.

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Section: Discussionmentioning

confidence: 74%

Section: In Vivo Two-photon Imaging Of Camp Dynamics In Mouse Cortexmentioning

confidence: 99%

A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging

Wang

Peng

et al. 2022

Preprint

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“…2). MSN excitability was persistently elevated for at least 4−5 min following dimaprit application, a feature that may indicate the involvement of PKA‐mediated protein phosphorylation (Gervasi et al., 2007; Ma et al., 2018). To test this hypothesis, we bath applied a cell‐permeable peptide inhibitor of PKA, PKI 14‐22 amide, myristoylated (PKI 1 μM), for at least 10 min prior to dimaprit application.…”

Section: Resultsmentioning

confidence: 99%

Activation of histamine type 2 receptors enhances intrinsic excitability of medium spiny neurons in the nucleus accumbens

Aceto

Nardella

Nanni

et al. 2022

The Journal of Physiology

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Histaminergic neurons are exclusively located in the hypothalamic tuberomammillary nucleus, from where they project to many brain areas including the nucleus accumbens (NAc), a brain area that integrates diverse monoaminergic inputs to coordinate motivated behaviours. While the NAc expresses various histamine receptor subtypes, the mechanisms by which histamine modulates NAc activity are still poorly understood. Using whole‐cell patch‐clamp recordings, we found that pharmacological activation of histamine 2 (H2) receptors elevates the excitability of NAc medium spiny neurons (MSNs), while activation of H1 receptors failed to significantly affect MSN excitability. The evoked firing of MSNs increased after seconds of local H2 agonist administration and remained elevated for minutes. H2 receptor (H2R) activation accelerated subthreshold depolarization in response to current injection, reduced the latency to fire, diminished action potential afterhyperpolarization and increased the action potential half‐width. The increased excitability was protein kinase A‐dependent and associated with decreased A‐type K+ currents. In addition, selective pharmacological inhibition of the Kv4.2 channel, the main molecular determinant of A‐type K+ currents in MSNs, mimicked and occluded the increased excitability induced by H2R activation. Our results indicate that histaminergic transmission in the NAc increases MSN intrinsic excitability through H2R‐dependent modulation of Kv4.2 channels. Activation of H2R will significantly alter spike firing in MSNs in vivo, and this effect could be an important mechanism by which these receptors mediate certain aspects of goal‐induced behaviours. Key points Histamine is synthesized and released by hypothalamic neurons of the tuberomammillary nucleus and serves as a general modulator for whole‐brain activity including the nucleus accumbens. Histamine receptors type 2 (HR2), which are expressed in the nucleus accumbens, couple to Gαs/off proteins which elevate cyclic adenosine monophosphate levels and activate protein kinase A. Whole‐cell patch‐clamp recordings revealed that H2R activation increased the evoked firing in medium spiny neurons of the nucleus accumbens via protein kinase A‐dependent mechanisms. HR2 activation accelerated subthreshold depolarization in response to current injection, reduced the latency to fire, diminished action potential medium after‐hyperpolarization and increased the action potential half‐width. HR2 activation also reduced A‐type potassium current. Selective pharmacological inhibition of the Kv4.2 channel mimicked and occluded the increased excitability induced by H2R activation.

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“…In addition, 2P FLIM has a relatively fast temporal resolution (∼1 s), has subcellular spatial resolution, and allows for easy manipulation of the sample within a microscope chamber. Recently, 2P lifetime imaging has been performed in vivo to measure molecular changes over time with single‐neuron resolution (Díaz‐García et al., 2017; Laviv et al., 2020; Ma et al., 2018). However, 2P FLIM also has disadvantages, such as the need to typically head‐restrain an animal as well as the complexity and cost of the equipment.…”

Section: Commentarymentioning

confidence: 99%

“…Such approaches may be sufficient to investigate long‐term and persistent molecular changes; however, more transient molecular changes in response to behavior will not be captured (Ramos et al., 2003). In vivo two‐photon (2P) fluorescence lifetime microscopy (FLIM) has been used for continuous imaging of biochemical state in vivo (Díaz‐García et al., 2017; Laviv et al., 2020; Ma et al., 2018), but this powerful approach is challenging, mainly applicable to analysis of superficial neurons, and restricted to head‐restrained behaviors. Therefore, to investigate the molecular changes during behavior in deep brain areas, we built on previous deep brain fluorescence‐based approaches (Cui et al., 2014; Goto et al., 2015) and developed single‐fiber fluorescence lifetime photometry (FLiP) (Lee, Chen, Lodder, & Sabatini, 2019, 2020), which allows for continuous lifetime monitoring while an animal is behaving.…”

Section: Introductionmentioning

confidence: 99%

Real‐Time, In Vivo Measurement of Protein Kinase A Activity in Deep Brain Structures Using Fluorescence Lifetime Photometry (FLiP)

2021

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The biochemical state of neurons, and of cells in general, is regulated by extracellular factors, including neurotransmitters, neuromodulators, and growth hormones. Interactions of an animal with its environment trigger neuromodulator release and engage biochemical transduction cascades to modulate synapse and cell function. Although these processes are thought to enact behavioral adaption to changing environments, when and where in the brain they are induced has been mysterious because of the challenge of monitoring biochemical state in real time in defined neurons in behaving animals. Here, we describe a method allowing measurement of activity of protein kinase A (PKA), an important intracellular effector for neuromodulators, in freely moving mice. To monitor PKA activity in vivo, we use a genetically targeted sensor (FLIM-AKAR) and fluorescence lifetime photometry (FLiP). This article describes how to set up a FLiP system and obtain robust recordings of net PKA phosphorylation state in vivo. The methods should be generally useful to monitor other pathways for which fluorescence lifetime reporters exist.

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A Highly Sensitive A-Kinase Activity Reporter for Imaging Neuromodulatory Events in Awake Mice

Cited by 6 publications

References 0 publications

A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging

A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging

Activation of histamine type 2 receptors enhances intrinsic excitability of medium spiny neurons in the nucleus accumbens

Real‐Time, In Vivo Measurement of Protein Kinase A Activity in Deep Brain Structures Using Fluorescence Lifetime Photometry (FLiP)

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