RuBi-Ruxolitinib: A Photoreleasable Antitumor JAK Inhibitor.

We describe the synthesis and biological testing of ruthenium-bipyridine ruxolitinib (RuBiRuxo), a photoreleasable form of ruxolitinib, a JAK inhibitor used as an antitumoral agent in cutaneous T-cell lymphomas (CTCL). This novel caged compound is synthesized efficiently, is stable in aqueous solution at room temperature, and is photoreleased rapidly by visible light. Irradiation of RuBiRuxo reduces cell proliferation and induces apoptosis in a light- and time-dependent manner in a CTCL cell line. This effect is specific and is mediated by a decreased phosphorylation of STAT proteins. Our results demonstrate the potential of ruthenium-based photocompounds and light-based therapeutic approaches for the potential treatment of cutaneous lymphomas and other pathologies.

Network-level encoding of local neurotransmitters in cortical astrocytes.

Astrocytes, the most abundant non-neuronal cell type in the mammalian brain, are crucial circuit components that respond to and modulate neuronal activity through calcium (Ca2+) signalling1-7. Astrocyte Ca2+ activity is highly heterogeneous and occurs across multiple spatiotemporal scales-from fast, subcellular activity3,4 to slow, synchronized activity across connected astrocyte networks8-10-to influence many processes5,7,11. However, the inputs that drive astrocyte network dynamics remain unclear. Here we used ex vivo and in vivo two-photon astrocyte imaging while mimicking neuronal neurotransmitter inputs at multiple spatiotemporal scales. We find that brief, subcellular inputs of GABA and glutamate lead to widespread, long-lasting astrocyte Ca2+ responses beyond an individual stimulated cell. Further, we find that a key subset of Ca2+ activity-propagative activity-differentiates astrocyte network responses to these two main neurotransmitters, and may influence responses to future inputs. Together, our results demonstrate that local, transient neurotransmitter inputs are encoded by broad cortical astrocyte networks over a minutes-long time course, contributing to accumulating evidence that substantial astrocyte-neuron communication occurs across slow, network-level spatiotemporal scales12-14. These findings will enable future studies to investigate the link between specific astrocyte Ca2+ activity and specific functional outputs, which could build a consistent framework for astrocytic modulation of neuronal activity.

Network-level encoding of local neurotransmitters in cortical astrocytes.

Astrocytes-the most abundant non-neuronal cell type in the mammalian brain-are crucial circuit components that respond to and modulate neuronal activity via calcium (Ca 2+ ) signaling 1-8 . Astrocyte Ca 2+ activity is highly heterogeneous and occurs across multiple spatiotemporal scales: from fast, subcellular activity 3,4 to slow, synchronized activity that travels across connected astrocyte networks 9-11 . Furthermore, astrocyte network activity has been shown to influence a wide range of processes 5,8,12 . While astrocyte network activity has important implications for neuronal circuit function, the inputs that drive astrocyte network dynamics remain unclear. Here we used ex vivo and in vivo two-photon Ca 2+ imaging of astrocytes while mimicking neuronal neurotransmitter inputs at multiple spatiotemporal scales. We find that brief, subcellular inputs of GABA and glutamate lead to widespread, long-lasting astrocyte Ca 2+ responses beyond an individual stimulated cell. Further, we find that a key subset of Ca 2+ activity-propagative events-differentiates astrocyte network responses to these two major neurotransmitters, and gates responses to future inputs. Together, our results demonstrate that local, transient neurotransmitter inputs are encoded by broad cortical astrocyte networks over the course of minutes, contributing to accumulating evidence across multiple model organisms that significant astrocyte-neuron communication occurs across slow, network-level spatiotemporal scales 13-15 . We anticipate that this study will be a starting point for future studies investigating the link between specific astrocyte Ca 2+ activity and specific astrocyte functional outputs, which could build a consistent framework for astrocytic modulation of neuronal activity.

A Photoactivatable Norepinephrine for Probing Adrenergic Neural Circuits.

Norepinephrine (NE) is a critical neuromodulator that mediates a wide range of behavior and neurophysiology, including attention, arousal, plasticity, and memory consolidation. A major source of NE is the brainstem nucleus the locus coeruleus (LC), which sends widespread projections throughout the central nervous system (CNS). Efforts to dissect this complex noradrenergic circuitry have driven the development of many tools that detect endogenous NE or modulate widespread NE release via LC activation and inhibition. While these tools have enabled research that elucidates physiological roles of NE, additional tools to probe these circuits with a higher degree of spatial precision could enable a finer delineation of function. Here, we describe the synthesis and chemical properties of a photo-activatable NE, [Ru(bpy) 2 (PMe 3 )(NE)]PF 6 (RuBi-NE). We validate the one-photon (1P) release of NE using whole-cell patch clamp electrophysiology in acute mouse brain slices containing the LC. We show that a 10 ms pulse of blue light, in the presence of RuBi-NE, briefly modulates the firing rate of LC neurons via α-2 adrenergic receptors. The development of a photo-activatable NE that can be released with light in the visible spectrum provides a new tool for fine-grained mapping of complex noradrenergic circuits, as well as the ability to probe how NE acts on non-neuronal cells in the CNS.

Shine, Shine, Ruthenium Caged Drug.

This is a highlight on the paper by Bonnet et al.: A Lock-and-Kill Anticancer Photoactivated Chemotherapy Agent. which constitutes an important step toward establishing photoactivated chemotherapy (PACT) as a widespread tool to treat different health issues, specially tumors. PACT can be a useful technique to deliver already tested drugs, where the effect of the desired molecule is directed only to its target after light irradiation, even in the cases in which it is difficult to achieve a precise delivery in the desired organ or tissue. Ruthenium-polipyridyl caged-compounds are near ideal devices to deliver a drug in that precise fashion, albeit they usually fail in revealing their actual location due to their weak light emission properties. The mentioned work introduces a simple and clever idea: the use of a covalently linked fluorophore to map the caged-compounds in-vivo distribution prior to the eventual irradiation to activate the chemotherapy.

Dopamine activates astrocytes in prefrontal cortex via α1-adrenergic receptors.

The prefrontal cortex (PFC) is a hub for cognitive control, and dopamine profoundly influences its functions. In other brain regions, astrocytes sense diverse neurotransmitters and neuromodulators and, in turn, orchestrate regulation of neuroactive substances. However, basic physiology of PFC astrocytes, including which neuromodulatory signals they respond to and how they contribute to PFC function, is unclear. Here, we characterize divergent signaling signatures in mouse astrocytes of the PFC and primary sensory cortex, which show differential responsiveness to locomotion. We find that PFC astrocytes express receptors for dopamine but are unresponsive through the Gs/Gi-cAMP pathway. Instead, fast calcium signals in PFC astrocytes are time locked to dopamine release and are mediated by α1-adrenergic receptors both ex vivo and in vivo. Further, we describe dopamine-triggered regulation of extracellular ATP at PFC astrocyte territories. Thus, we identify astrocytes as active players in dopaminergic signaling in the PFC, contributing to PFC function though neuromodulator receptor crosstalk.

Active Surveillance of Asymptomatic, Presymptomatic, and Oligosymptomatic SARS-CoV-2-Infected Individuals in Communities Inhabiting Closed or Semi-closed Institutions.

Background: The high COVID-19 dissemination rate demands active surveillance to identify asymptomatic, presymptomatic, and oligosymptomatic (APO) SARS-CoV-2-infected individuals. This is of special importance in communities inhabiting closed or semi-closed institutions such as residential care homes, prisons, neuropsychiatric hospitals, etc., where risk people are in close contact. Thus, a pooling approach-where samples are mixed and tested as single pools-is an attractive strategy to rapidly detect APO-infected in these epidemiological scenarios. Materials and Methods: This study was done at different pandemic periods between May 28 and August 31 2020 in 153 closed or semi-closed institutions in the Province of Buenos Aires (Argentina). We setup pooling strategy in two stages: first a pool-testing followed by selective individual-testing according to pool results. Samples included in negative pools were presumed as negative, while samples from positive pools were re-tested individually for positives identification. Results: Sensitivity in 5-sample or 10-sample pools was adequate since only 2 Ct values were increased with regard to single tests on average. Concordance between 5-sample or 10-sample pools and individual-testing was 100% in the Ct ≤ 36. We tested 4,936 APO clinical samples in 822 pools, requiring 86-50% fewer tests in low-to-moderate prevalence settings compared to individual testing. Conclusions: By this strategy we detected three COVID-19 outbreaks at early stages in these institutions, helping to their containment and increasing the likelihood of saving lives in such places where risk groups are concentrated.

Cis-Trans Interconversion in Ruthenium(II) Bipyridine Complexes.

Most studies of ruthenium polypyridine complexes are devoted to their cis isomers. The fact that cis isomers are thermally more stable and thus easier to synthesize has prevented researchers from investigating the properties and applications of trans complexes. We present a study of thermal and photochemical cis-trans interconversion of the key complex [Ru(bpy)2(PMe3)(H2O)]2+ (bpy = 2,2'-bipyridine, PMe3 = trimethylphosphine), which results in specific synthetic applications of the trans species, potentially useful as a platform for designing highly efficient visible light activated caged compounds. We show, as a proof of concept, some examples of trans complexes bearing N-donor and P-donor ligands and their comparison with the cis isomers.

Direct Measurement of Two-Photon Action Cross Section.

We present a simple and fast methodology for measuring the two-photon (2P) action cross section of phototriggers. The method uses a standard 2P microscopy setup for both uncaging and detection and a set of lithographically made microcuvettes in order to reduce the total excitation volume and, thus, the photolysis time. The procedure does not need a standard and can be used for any caged compounds that present different emission properties before and after uncaging. We tested the method with 2P active ruthenium-based caged serotonin and compared the obtained value with a standard measure involving fluorescein as reference.

Optical manipulation of animal behavior using a ruthenium-based phototrigger.

A visible-light activatable caged compound based on a ruthenium-polypyridine complex was used to elicit the feeding response of the freshwater cnidarian Hydra vulgaris. The phototrigger delivers l-arginine in a clean reaction under irradiation with blue or green light. The synthesis, characterization and application mode of this caged arginine are described. A combination of fiber-optics setup and a high absorbance medium allows the precise control of uncaging in the submillimetric range, needed to address the zone where activation takes place.

Multiway analysis through direct excitation-emission matrix imaging.

In this work, a direct in-flow methodology for the acquisition of excitation-emission fluorescence matrices is presented. The system is particularly suited for measurements in the order of tens of milliseconds. A light source operated in continuous mode is dispersed through a grating and focused onto a square-section capillary. Under the spatially resolved excitation, the emission is collected, dispersed through a second grating and further focused onto a CCD array sensor. To allow the wavelength accuracy, a spectral calibration was performed registering the scattering signal of a dispersive element using interference filters ranging from 340 nm to 740 nm. The theoretical performance of the method was analyzed and second-order data obtained for different analyte mixtures are presented and discussed. PARAFAC was applied to evaluate the trilinearity of the obtained data. Mathematical evaluation by means of the criterion of similarity corroborates the agreement between experimental pure spectra and spectral profiles retrieved from PARAFAC. Moreover, the feasibility of the spectrometer to obtain second-order data for analyses with quantitative aims was demonstrated. Finally, fast data acquisition was proved by monitoring a chromatographic analysis of dye mixtures for the generation of third-order LC-EEM data. Here, an improvement in the resolution of the different instrumental modes was demonstrated.

Inhibition of striatal cholinergic interneuron activity by the Kv7 opener retigabine and the nonsteroidal anti-inflammatory drug diclofenac.

Striatal cholinergic interneurons provide modulation to striatal circuits involved in voluntary motor control and goal-directed behaviors through their autonomous tonic discharge and their firing "pause" responses to novel and rewarding environmental events. Striatal cholinergic interneuron hyperactivity was linked to the motor deficits associated with Parkinson's disease and the adverse effects of chronic antiparkinsonian therapy like l-DOPA-induced dyskinesia. Here we addressed whether Kv7 channels, which provide negative feedback to excitation in other neuron types, are involved in the control of striatal cholinergic interneuron tonic activity and response to excitatory inputs. We found that autonomous firing of striatal cholinergic interneurons is not regulated by Kv7 channels. In contrast, Kv7 channels limit the summation of excitatory postsynaptic potentials in cholinergic interneurons through a postsynaptic mechanism. Striatal cholinergic interneurons have a high reserve of Kv7 channels, as their opening using pharmacological tools completely silenced the tonic firing and markedly reduced their intrinsic excitability. A strong inhibition of striatal cholinergic interneurons was also observed in response to the anti-inflammatory drugs diclofenac and meclofenamic acid, however, this effect was independent of Kv7 channels. These data bring attention to new potential molecular targets and pharmacological tools to control striatal cholinergic interneuron activity in pathological conditions where they are believed to be hyperactive, including Parkinson's disease.

Dopamine Modulates Delta-Gamma Phase-Amplitude Coupling in the Prefrontal Cortex of Behaving Rats.

Dopamine release and phase-amplitude cross-frequency coupling (CFC) have independently been implicated in prefrontal cortex (PFC) functioning. To causally investigate whether dopamine release affects phase-amplitude comodulation between different frequencies in local field potentials (LFP) recorded from the medial PFC (mPFC) of behaving rats, we used RuBiDopa, a light-sensitive caged compound that releases the neurotransmitter dopamine when irradiated with visible light. LFP power did not change in any frequency band after the application of light-uncaged dopamine, but significantly strengthened phase-amplitude comodulation between delta and gamma oscillations. Saline did not exert significant changes, while injections of dopamine and RuBiDopa produced a slow increase in comodulation for several minutes after the injection. The results show that dopamine release in the medial PFC shifts phase-amplitude comodulation from theta-gamma to delta-gamma. Although being preliminary results due to the limitation of the low number of animals present in this study, our findings suggest that dopamine-mediated modification of the frequencies involved in comodulation could be a mechanism by which this neurotransmitter regulates functioning in mPFC.

A Visible-Light-Sensitive Caged Serotonin.

Serotonin, or 5-hydroxytryptamine (5HT), is an important neurotransmitter in the nervous system of both vertebrates and invertebrates. Deficits in 5HT signaling are responsible for many disabling psychiatric conditions, and its molecular machinery is the target of many pharmaceuticals. We present a new 5HT phototrigger, the compound [Ru(bpy)2(PMe3)(5HT)]2+, where PMe3 is trimethylphosphine. As with other ruthenium-bipyridyl based caged compounds, [Ru(bpy)2(PMe3)(5HT)]2+ presents activity in the visible region of the spectrum. We characterize and discuss the photochemical properties of the caged compound, and demonstrate its use by modulating the excitability of mouse prefrontal principal neurons.

Manipulating pH using near-infrared light assisted by upconverting nanoparticles.

Near-infrared light can be used to manipulate the pH of aqueous solutions by using upconverting nanoparticle-assisted photocleavage of a ruthenium complex photobase. Upconverting nanoparticles and the photobase were also introduced into a pH-responsive hydrogel, in which near-infrared irradiation induced swelling of the hydrogel.

Multiphoton Excitation of Upconverting Nanoparticles in Pulsed Regime.

Upconverting nanoparticles (UCNPs) present emission in the visible region upon irradiation with NIR light through a multiphoton mechanism. However, the long characteristic time of their emission has prevented the use of this kind of entities as multiphoton probes. We present a study on the use of erbium-containing UCNPs under pulsed excitation, showing that both the power density and the duration of the excitation pulse are key factors to understand the emission behavior. By adjusting power and excitation rate, we can obtain typical multiphoton z-axis focal exclusive excitation. These findings open the possibility of using UCNPs as probes for controlled localization of uncaging and imaging with multiphoton z-axis sectioning. We show that this can be achieved even at power densities several orders of magnitude lower than traditional multiphoton microscopies.

Chemical two-photon fluorescence.

We describe a method based on a caged fluorescent molecule that can act as a chemical two-photon probe. It is composed of an organic fluorophore and a ruthenium-bipyridine complex that acts as a photoremovable quencher. For the fluorophore to be emissive, two independent photons must act on the molecule: the first photon frees the fluorescent ligand from the Ru complex and the second photon excites the fluorescence. In this two-photon regime, the emission is not proportional to the excitation intensity but rather to its second power, as in traditional two-photon systems based on ultrashort pulsed high-power lasers. This quadratic relationship implies a much higher spatial precision on the z-axis when the probe is used in a microscopy technique. The chemical nature of the two-photon excitation mechanism allows the use of inexpensive low-power lasers.

Fluorescent ligands and energy transfer in photoactive ruthenium-bipyridine complexes.

Ruthenium bis(bipyridine) complexes have proved to be useful as phototriggers for visible and IR-light photodelivery of molecules. They usually expel one ligand heterolytically upon absorption of blue or green light. However, their absorption capabilities at wavelengths longer than 500 nm are poor. Through coordination of fluorescent ligands to the Ru center, it is possible to establish an energy transfer pathway that allows these kinds of complexes to extend the range of photoactivation up to yellow wavelengths. We introduce a study of this effect in several complexes of the family using a modified Rhodamine as fluorescent ligand with different coordinated linkers. The observed trends show that a rational design of fluorophore-enhanced Ru-bpy phototriggers is possible and that photolysis efficiency can be increased by choosing the right combination of ligands.

Ruthenium polypyridyl phototriggers: from beginnings to perspectives.

Octahedral Ru(II) polypyridyl complexes constitute a superb platform to devise photoactive triggers capable of delivering entire molecules in a reliable, fast, efficient and clean way. Ruthenium coordination chemistry opens the way to caging a wide range of molecules, such as amino acids, nucleotides, neurotransmitters, fluorescent probes and genetic inducers. Contrary to other phototriggers, these Ru-based caged compounds are active with visible light, and can be photolysed even at 532 nm (green), enabling the use of simple and inexpensive equipment. These compounds are also active in the two-photon regime, a property that extends their scope to systems where IR light must be used to achieve high precision and penetrability. The state of the art and the future of ruthenium polypyridyl phototriggers are discussed, and several new applications are presented.