Fast and Artifact-Free Excitation Multiplexing Using Synchronized Image Scanning.

Multicolor fluorescence microscopy is an essential tool to visualize structures and dynamics in the life and materials sciences. However, the near-simultaneous acquisition of labels differing in excitation spectrum is difficult and renders such measurements prone to artifacts. We present a simple strategy to provide quasi-simultaneous fluorescence imaging with multiple excitation wavelengths by using an optical element to displace the sample image on the sensor at a rate that is much faster than the image acquisition rate and synchronizing this with the illumination. The emission elicited by the different wavelengths can then be encoded into the point-spread function of the imaging or visualized as multiple distinct images. In doing so, our approach can eliminate or mitigate artifacts caused by temporal aliasing in conventional sequential imaging. We demonstrate the use of our system to uncover hidden emissive states in single quantum dots and for the imaging of Ca2+ signaling in neurons.

Fluorescent biosensor imaging meets deterministic mathematical modelling: quantitative investigation of signalling compartmentalization.

Cells execute specific responses to diverse environmental cues by encoding information in distinctly compartmentalized biochemical signalling reactions. Genetically encoded fluorescent biosensors enable the spatial and temporal monitoring of signalling events in live cells. Temporal and spatiotemporal computational models can be used to interpret biosensor experiments in complex biochemical networks and to explore hypotheses that are difficult to test experimentally. In this review, we first provide brief discussions of the experimental toolkit of fluorescent biosensors as well as computational basics with a focus on temporal and spatiotemporal deterministic models. We then describe how we used this combined approach to identify and investigate a protein kinase A (PKA) - cAMP - Ca2+ oscillatory circuit in MIN6 ß cells, a mouse pancreatic ß cell system. We describe the application of this combined approach to interrogate how this oscillatory circuit is differentially regulated in a nano-compartment formed at the plasma membrane by the scaffolding protein A kinase anchoring protein 79/150. We leveraged both temporal and spatiotemporal deterministic models to identify the key regulators of this oscillatory circuit, which we confirmed with further experiments. The powerful approach of combining live-cell biosensor imaging with quantitative modelling, as discussed here, should find widespread use in the investigation of spatiotemporal regulation of cell signalling.

CODEX, a neural network approach to explore signaling dynamics landscapes.

Current studies of cell signaling dynamics that use live cell fluorescent biosensors routinely yield thousands of single-cell, heterogeneous, multi-dimensional trajectories. Typically, the extraction of relevant information from time series data relies on predefined, human-interpretable features. Without a priori knowledge of the system, the predefined features may fail to cover the entire spectrum of dynamics. Here we present CODEX, a data-driven approach based on convolutional neural networks (CNNs) that identifies patterns in time series. It does not require a priori information about the biological system and the insights into the data are built through explanations of the CNNs' predictions. CODEX provides several views of the data: visualization of all the single-cell trajectories in a low-dimensional space, identification of prototypic trajectories, and extraction of distinctive motifs. We demonstrate how CODEX can provide new insights into ERK and Akt signaling in response to various growth factors, and we recapitulate findings in p53 and TGFß-SMAD2 signaling.

Switch-like PKA responses in the nucleus of striatal neurons.

Although it is known that protein kinase A (PKA) in the nucleus regulates gene expression, the specificities of nuclear PKA signaling remain poorly understood. Here, we combined computational modeling and live-cell imaging of PKA-dependent phosphorylation in mouse brain slices to investigate how transient dopamine signals are translated into nuclear PKA activity in cortical pyramidal neurons and striatal medium spiny neurons. We observed that the nuclear PKA signal in striatal neurons featured an ultrasensitive responsiveness, associated with fast all-or-none responses, which is not consistent with the commonly accepted theory of a slow and passive diffusion of catalytic PKA in the nucleus. Our numerical model suggests that a positive feed-forward mechanism inhibiting nuclear phosphatase activity - possibly mediated by DARPP-32 (also known as PPP1R1B) - could be responsible for this non-linear pattern of nuclear PKA response, allowing for a better detection of the transient dopamine signals that are often associated with reward-mediated learning.

Phosphodiesterase 1 Bridges Glutamate Inputs with NO- and Dopamine-Induced Cyclic Nucleotide Signals in the Striatum.

The calcium-regulated phosphodiesterase 1 (PDE1) family is highly expressed in the brain, but its functional role in neurones is poorly understood. Using the selective PDE1 inhibitor Lu AF64196 and biosensors for cyclic nucleotides including a novel biosensor for cGMP, we analyzed the effect of PDE1 on cAMP and cGMP in individual neurones in brain slices from male newborn mice. Release of caged NMDA triggered a transient increase of intracellular calcium, which was associated with a decrease in cAMP and cGMP in medium spiny neurones in the striatum. Lu AF64196 alone did not increase neuronal cyclic nucleotide levels, but blocked the NMDA-induced reduction in cyclic nucleotides indicating that this was mediated by calcium-activated PDE1. Similar effects were observed in the prefrontal cortex and the hippocampus. Upon corelease of dopamine and NMDA, PDE1 was shown to down-regulate the D1-receptor mediated increase in cAMP. PDE1 inhibition increased long-term potentiation in rat ventral striatum, showing that PDE1 is implicated in the regulation of synaptic plasticity. Overall, our results show that PDE1 reduces cyclic nucleotide signaling in the context of glutamate and dopamine coincidence. This effect could have a therapeutic value for treating brain disorders related to dysfunctions in dopamine neuromodulation.

Detection of phasic dopamine by D1 and D2 striatal medium spiny neurons.

KEY POINTS: Brief dopamine events are critical actors of reward-mediated learning in the striatum; the intracellular cAMP-protein kinase A (PKA) response of striatal medium spiny neurons to such events was studied dynamically using a combination of biosensor imaging in mouse brain slices and in silico simulations. Both D1 and D2 medium spiny neurons can sense brief dopamine transients in the sub-micromolar range. While dopamine transients profoundly change cAMP levels in both types of medium spiny neurons, the PKA-dependent phosphorylation level remains unaffected in D2 neurons. At the level of PKA-dependent phosphorylation, D2 unresponsiveness depends on protein phosphatase-1 (PP1) inhibition by DARPP-32. Simulations suggest that D2 medium spiny neurons could detect transient dips in dopamine level. ABSTRACT: The phasic release of dopamine in the striatum determines various aspects of reward and action selection, but the dynamics of the dopamine effect on intracellular signalling remains poorly understood. We used genetically encoded FRET biosensors in striatal brain slices to quantify the effect of transient dopamine on cAMP or PKA-dependent phosphorylation levels, and computational modelling to further explore the dynamics of this signalling pathway. Medium-sized spiny neurons (MSNs), which express either D1 or D2 dopamine receptors, responded to dopamine by an increase or a decrease in cAMP, respectively. Transient dopamine showed similar sub-micromolar efficacies on cAMP in both D1 and D2 MSNs, thus challenging the commonly accepted notion that dopamine efficacy is much higher on D2 than on D1 receptors. However, in D2 MSNs, the large decrease in cAMP level triggered by transient dopamine did not translate to a decrease in PKA-dependent phosphorylation level, owing to the efficient inhibition of protein phosphatase 1 by DARPP-32. Simulations further suggested that D2 MSNs can also operate in a 'tone-sensing' mode, allowing them to detect transient dips in basal dopamine. Overall, our results show that D2 MSNs may sense much more complex patterns of dopamine than previously thought.

In Vivo Two-Photon Microscopy Reveals Sensory-Evoked Serotonin (5-HT) Release in Adult Mammalian Neocortex.

The recent development of genetically encoded fluorescent neurotransmitter biosensors has opened the door to recording serotonin (5-hydroxytryptamine, 5-HT) signaling dynamics with high temporal and spatial resolution in vivo. While this represents a significant step forward for serotonin research, the utility of available 5-HT biosensors remains to be fully established under diverse in vivo conditions. Here, we used two-photon microscopy in awake mice to examine the effectiveness of specific 5-HT biosensors for monitoring 5-HT dynamics in somatosensory cortex. Initial experiments found that whisker stimulation evoked a striking change in 5-HT biosensor signal. However, similar changes were observed in controls expressing green fluorescent protein, suggesting a potential hemodynamic artifact. Subsequent use of a second control fluorophore with emission peaks separated from the 5-HT biosensor revealed a reproducible, stimulus-locked increase in 5-HT signal. Our data highlight the promise of 5-HT biosensors for in vivo application, provided measurements are carried out with appropriate optical controls.

Transient cAMP production drives rapid and sustained spiking in brainstem parabrachial neurons to suppress feeding.

Brief stimuli can trigger longer-lasting brain states. G-protein-coupled receptors (GPCRs) could help sustain such states by coupling slow-timescale molecular signals to neuronal excitability. Brainstem parabrachial nucleus glutamatergic (PBNGlut) neurons regulate sustained brain states such as pain and express Gs-coupled GPCRs that increase cAMP signaling. We asked whether cAMP in PBNGlut neurons directly influences their excitability and effects on behavior. Both brief tail shocks and brief optogenetic stimulation of cAMP production in PBNGlut neurons drove minutes-long suppression of feeding. This suppression matched the duration of prolonged elevations in cAMP, protein kinase A (PKA) activity, and calcium activity in vivo and ex vivo, as well as sustained, PKA-dependent increases in action potential firing ex vivo. Shortening this elevation in cAMP reduced the duration of feeding suppression following tail shocks. Thus, molecular signaling in PBNGlut neurons helps prolong neural activity and behavioral states evoked by brief, salient bodily stimuli.

Brain-targeting, acid-responsive antioxidant nanoparticles for stroke treatment and drug delivery.

Stroke is the leading cause of death and disability. Currently, there is no effective pharmacological treatment for this disease, which can be partially attributed to the inability to efficiently deliver therapeutics to the brain. Here we report the development of natural compound-derived nanoparticles (NPs), which function both as a potent therapeutic agent for stroke treatment and as an efficient carrier for drug delivery to the ischemic brain. First, we screened a collection of natural nanomaterials and identified betulinic acid (BA) as one of the most potent antioxidants for stroke treatment. Next, we engineered BA NPs for preferential drug release in acidic ischemic tissue through chemically converting BA to betulinic amine (BAM) and for targeted drug delivery through surface conjugation of AMD3100, a CXCR4 antagonist. The resulting AMD3100-conjugated BAM NPs, or A-BAM NPs, were then assessed as a therapeutic agent for stroke treatment and as a carrier for delivery of NA1, a neuroprotective peptide. We show that intravenous administration of A-BAM NPs effectively improved recovery from stroke and its efficacy was further enhanced when NA1 was encapsulated. Due to their multifunctionality and significant efficacy, we anticipate that A-BAM NPs have the potential to be translated both as a therapeutic agent and as a drug carrier to improve the treatment of stroke.

Cellular context shapes cyclic nucleotide signaling in neurons through multiple levels of integration.

Intracellular signaling with cyclic nucleotides are ubiquitous signaling pathways, yet the dynamics of these signals profoundly differ in different cell types. Biosensor imaging experiments, by providing direct measurements in intact cellular environment, reveal which receptors are activated by neuromodulators and how the coincidence of different neuromodulators is integrated at various levels in the signaling cascade. Phosphodiesterases appear as one important determinant of cross-talk between different signaling pathways. Finally, analysis of signal dynamics reveal that striatal medium-sized spiny neuron obey a different logic than other brain regions such as cortex, probably in relation with the function of this brain region which efficiently detects transient dopamine.

CDK2 limits the highly energetic secretory program of mature ß cells by restricting PEP cycle-dependent KATP channel closure.

Hallmarks of mature ß cells are restricted proliferation and a highly energetic secretory state. Paradoxically, cyclin-dependent kinase 2 (CDK2) is synthesized throughout adulthood, its cytosolic localization raising the likelihood of cell cycle-independent functions. In the absence of any changes in ß cell mass, maturity, or proliferation, genetic deletion of Cdk2 in adult ß cells enhanced insulin secretion from isolated islets and improved glucose tolerance in vivo. At the single ß cell level, CDK2 restricts insulin secretion by increasing KATP conductance, raising the set point for membrane depolarization in response to activation of the phosphoenolpyruvate (PEP) cycle with mitochondrial fuels. In parallel with reduced ß cell recruitment, CDK2 restricts oxidative glucose metabolism while promoting glucose-dependent amplification of insulin secretion. This study provides evidence of essential, non-canonical functions of CDK2 in the secretory pathways of quiescent ß cells.

Pyruvate Kinase Controls Signal Strength in the Insulin Secretory Pathway.

Pancreatic ß cells couple nutrient metabolism with appropriate insulin secretion. Here, we show that pyruvate kinase (PK), which converts ADP and phosphoenolpyruvate (PEP) into ATP and pyruvate, underlies ß cell sensing of both glycolytic and mitochondrial fuels. Plasma membrane-localized PK is sufficient to close KATP channels and initiate calcium influx. Small-molecule PK activators increase the frequency of ATP/ADP and calcium oscillations and potently amplify insulin secretion. PK restricts respiration by cyclically depriving mitochondria of ADP, which accelerates PEP cycling until membrane depolarization restores ADP and oxidative phosphorylation. Our findings support a compartmentalized model of ß cell metabolism in which PK locally generates the ATP/ADP required for insulin secretion. Oscillatory PK activity allows mitochondria to perform synthetic and oxidative functions without any net impact on glucose oxidation. These findings suggest a potential therapeutic route for diabetes based on PK activation that would not be predicted by the current consensus single-state model of ß cell function.

Imaging Intracellular H2O2 with the Genetically Encoded PerFRET and OxyFRET Probes.

Hydrogen peroxide (H2O2) is an important signaling intermediate with various regulatory and effector functions. Despite its significance, the subcellular organization of H2O2 signals is poorly understood. Introducing novel techniques for the intracellular detection of H2O2 would be essential for a more complete understanding of its role in cellular signaling. We previously reported the development of two novel fluorescence resonance energy transfer (FRET)-based protein sensors that showed opposite emission ratio changes upon reaction with H2O2. In this chapter, we detail the methods for using OxyFRET and PerFRET for the assessment of changes in subcellular H2O2 levels.

The high efficacy of muscarinic M4 receptor in D1 medium spiny neurons reverses striatal hyperdopaminergia.

The opposing action of dopamine and acetylcholine has long been known to play an important role in basal ganglia physiology. However, the quantitative analysis of dopamine and acetylcholine signal interaction has been difficult to perform in the native context because the striatum comprises mainly two subtypes of medium-sized spiny neurons (MSNs) on which these neuromodulators exert different actions. We used biosensor imaging in live brain slices of dorsomedial striatum to monitor changes in intracellular cAMP at the level of individual MSNs. We observed that the muscarinic agonist oxotremorine decreases cAMP selectively in the MSN subpopulation that also expresses D1 dopamine receptors, an action mediated by the M4 muscarinic receptor. This receptor has a high efficacy on cAMP signaling and can shut down the positive cAMP response induced by dopamine, at acetylcholine concentrations which are consistent with physiological levels. This supports our prediction based on theoretical modeling that acetylcholine could exert a tonic inhibition on striatal cAMP signaling, thus supporting the possibility that a pause in acetylcholine release is required for phasic dopamine to transduce a cAMP signal in D1 MSNs. In vivo experiments with acetylcholinesterase inhibitors donepezil and tacrine, as well as with the positive allosteric modulators of M4 receptor VU0152100 and VU0010010 show that this effect is sufficient to reverse the increased locomotor activity of DAT-knockout mice. This suggests that M4 receptors could be a novel therapeutic target to treat hyperactivity disorders.

Selective Effects of PDE10A Inhibitors on Striatopallidal Neurons Require Phosphatase Inhibition by DARPP-32

Type 10A phosphodiesterase (PDE10A) is highly expressed in the striatum, in striatonigral and striatopallidal medium-sized spiny neurons (MSNs), which express D1 and D2 dopamine receptors, respectively. PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear. We analyzed the effects of PDE10A inhibition in vivo by immunohistochemistry, and imaged cAMP, cAMP-dependent protein kinase A (PKA), and cGMP signals with biosensors in mouse brain slices. PDE10A inhibition in mouse striatal slices produced a steady-state increase in intracellular cAMP concentration in D1 and D2 MSNs, demonstrating that PDE10A regulates basal cAMP levels. Surprisingly, the PKA-dependent AKAR3 phosphorylation signal was strong in D2 MSNs, whereas D1 MSNs remained unresponsive. This effect was also observed in adult mice in vivo since PDE10A inhibition increased phospho-histone H3 immunoreactivity selectively in D2 MSNs in the dorsomedial striatum. The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition. These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs. This study shows that PDE10A inhibitors share with antipsychotic medications the property of activating preferentially PKA-dependent signaling in D2 MSNs.

The NO/cGMP pathway inhibits transient cAMP signals through the activation of PDE2 in striatal neurons.

The NO-cGMP signaling plays an important role in the regulation of striatal function although the mechanisms of action of cGMP specifically in medium spiny neurons (MSNs) remain unclear. Using genetically encoded fluorescent biosensors, including a novel Epac-based sensor (EPAC-S(H150)) with increased sensitivity for cAMP, we analyze the cGMP response to NO and whether it affected cAMP/PKA signaling in MSNs. The Cygnet2 sensor for cGMP reported large responses to NO donors in both striatonigral and striatopallidal MSNs, this cGMP signal was controlled partially by PDE2. At the level of cAMP brief forskolin stimulations produced transient cAMP signals which differed between D1 and D2 MSNs. NO inhibited these cAMP transients through cGMP-dependent PDE2 activation, an effect that was translated and magnified downstream of cAMP, at the level of PKA. PDE2 thus appears as a critical effector of NO which modulates the post-synaptic response of MSNs to dopaminergic transmission.