Contributions of the Sodium Leak Channel NALCN to Pacemaking of Medial Ventral Tegmental Area and Substantia Nigra Dopaminergic Neurons.

We tested the role of the sodium leak channel, NALCN, in pacemaking of dopaminergic neuron (DAN) subpopulations from adult male and female mice. In situ hybridization revealed NALCN RNA in all DANs, with lower abundance in medial ventral tegmental area (VTA) relative to substantia nigra pars compacta (SNc). Despite lower relative abundance of NALCN, we found that acute pharmacological blockade of NALCN in medial VTA DANs slowed pacemaking by 49.08%. We also examined the electrophysiological properties of projection-defined VTA DAN subpopulations identified by retrograde labeling. Inhibition of NALCN reduced pacemaking in DANs projecting to medial nucleus accumbens (NAc) and others projecting to lateral NAc by 70.74% and 31.98%, respectively, suggesting that NALCN is a primary driver of pacemaking in VTA DANs. In SNc DANs, potentiating NALCN by lowering extracellular calcium concentration speeded pacemaking in wildtype but not NALCN conditional knockout mice, demonstrating functional presence of NALCN. In contrast to VTA DANs, however, pacemaking in SNc DANs was unaffected by inhibition of NALCN. Instead, we found that inhibition of NALCN increased the gain of frequency-current plots at firing frequencies slower than spontaneous firing. Similarly, inhibition of the hyperpolarization-activated cyclic nucleotide-gated (HCN) conductance increased gain but had little effect on pacemaking. Interestingly, simultaneous inhibition of NALCN and HCN resulted in significant reduction in pacemaker rate. Thus, we found NALCN makes substantial contributions to driving pacemaking in VTA DAN subpopulations. In SNc DANs, NALCN is not critical for pacemaking but inhibition of NALCN makes cells more sensitive to hyperpolarizing stimuli.SIGNIFICANCE STATEMENT Pacemaking in midbrain dopaminergic neurons (DAN) relies on multiple subthreshold conductances, including a sodium leak. Whether the sodium leak channel, NALCN, contributes to pacemaking in DANs located in the VTA and the SNc has not yet been determined. Using electrophysiology and pharmacology, we show that NALCN plays a prominent role in driving pacemaking in projection-defined VTA DAN subpopulations. By contrast, pacemaking in SNc neurons does not rely on NALCN. Instead, the presence of NALCN regulates the excitability of SNc DANs by reducing the gain of the neuron's response to inhibitory stimuli. Together, these findings will inform future efforts to obtain DAN subpopulation-specific treatments for use in neuropsychiatric disorders.

Bidirectional Neuronal Actuation by Uncaging with Violet and Green Light.

We have developed a photochemical protecting group that enables wavelength selective uncaging using green versus violet light. Change of the exocyclic oxygen of the laser dye coumarin-102 to sulfur, gave thio-coumarin-102, a new chromophore with an absorption ratio at 503/402 nm of 37. Photolysis of thio-coumarin-102 caged γ-aminobutyric acid was found to be highly wavelength selective on neurons, with normalized electrical responses >100-fold higher in the green versus violet channel. When partnered with coumarin-102 caged glutamate, we could use whole cell violet and green irradiation to fire and block neuronal action potentials with complete orthogonality. Localized irradiation of different dendritic segments, each connected to a neuronal cell body, in concert with 3-dimenional Ca2+ imaging, revealed that such inputs could function independently. Chemical signaling in living cells always involves a complex balance of multiple pathways, use of (thio)-coumarin-102 caged compounds will enable arbitrarily timed flashes of green and violet light to interrogate two independent pathways simultaneously.

Photopotentiation of the GABAA receptor with caged diazepam.

As the inhibitory γ-aminobutyric acid-ergic (GABAergic) transmission has a pivotal role in the central nervous system (CNS) and defective forms of its synapses are associated with serious neurological disorders, numerous versions of caged GABA and, more recently, photoswitchable ligands have been developed to investigate such transmission. While the complementary nature of these probes is evident, the mechanisms by which the GABA receptors can be photocontrolled have not been fully exploited. In fact, the ultimate need for specificity is critical for the proper synaptic exploration. No caged allosteric modulators of the GABAA receptor have been reported so far; to introduce such an investigational approach, we exploited the structural motifs of the benzodiazepinic scaffold to develop a photocaged version of diazepam (CD) that was tested on basolateral amygdala (BLa) pyramidal cells in mouse brain slices. CD is devoid of any intrinsic activity toward the GABAA receptor before irradiation. Importantly, CD is a photoreleasable GABAA receptor-positive allosteric modulator that offers a different probing mechanism compared to caged GABA and photoswitchable ligands. CD potentiates the inhibitory signaling by prolonging the decay time of postsynaptic GABAergic currents upon photoactivation. Additionally, no effect on presynaptic GABA release was recorded. We developed a photochemical technology to individually study the GABAA receptor, which specifically expands the toolbox available to study GABAergic synapses.

Chemical tuning of photoswitchable azobenzenes: a photopharmacological case study using nicotinic transmission.

We have developed photochromic probes for the nicotinic acetylcholine receptor that exploit the unique chemical properties of the tetrafluoroazobenzene (4FAB) scaffold. Ultraviolet light switching and rapid thermal relaxation of the metastable cis configuration are the main drawbacks associated with standard AB-based switches. We designed our photoprobes to take advantage of the excellent thermodynamic stability of the cis-4FAB configuration (thermal half-life > 12 days at 37 °C in physiological buffer) and cis-trans photostationary states above 84%. Furthermore, the well-separated n-π* absorption bands of trans- and cis-4FAB allow facile photoswitching with visible light in two optical channels. A convergent 11-step synthetic approach allowed the installation of a trimethylammonium (TA) head onto the 4FAB scaffold, by means of an alkyl spacer, to afford a free diffusible 4FABTA probe. TAs are known to agonize nicotinic receptors, so 4FABTA was tested on mouse brain slices and enabled reversible receptor activation with cycles of violet and green light. Due to the very long-lived metastable cis configuration, 4FAB in vivo use could be of great promise for long term biological studies. Further chemical functionalization of this 4FAB probe with a maleimide functionality allowed clean cross-linking with glutathione. However, attempts to conjugate with a cysteine on a genetically modified nicotinic acetylcholine receptor did not afford the expected light-responsive channel. Our data indicate that the 4FAB photoswitch can be derivatized bifunctionally for genetically-targeted photopharmacology whilst preserving all the favorable photophysical properties of the parent 4FAB scaffold, however, the tetrafluoro motif can significantly perturb pharmacophore-protein interactions. In contrast, we found that the freely diffusible 4FABTA probe could be pre-set with green light into an OFF state that was biologically inert, irradiation with violet light effectively "uncaged" agonist activity, but in a photoreversible manner. Since the neurotransmitter acetylcholine has fully saturated heteroatom valences, our photoswitchable 4FABTA probe could be useful for physiological studies of this neurotransmitter.

A Photoswitchable Fluorophore for the Real-Time Monitoring of Dynamic Events in Living Organisms.

This study reports the synthesis of a photoactivatable fluorophore with optimal photochemical and photophysical properties for the real-time tracking of motion in vivo. The photoactivation mechanism designed into this particular compound permits the conversion of an emissive reactant into an emissive product with resolved fluorescence, under mild illumination conditions that are impossible to replicate with conventional switching schemes based on bleaching. Indeed, the supramolecular delivery of these photoswitchable probes into the cellular blastoderm of Drosophila melanogaster embryos allows the real-time visualization of translocating molecules with no detrimental effects on the developing organisms. Thus, this innovative mechanism for fluorescence photoactivation can evolve into a general chemical tool to monitor dynamic processes in living biological specimens.

High-Throughput Single-Molecule Spectroscopy Resolves the Conformational Isomers of BODIPY Chromophores.

A borondipyrromethene (BODIPY) chromophore is connected to a benzoxazole, benzothiazole, or nitrobenzothiazole heterocycle through an olefinic bridge with trans configuration. Rotation about the two [C-C] bonds flanking the olefinic bridge occurs with fast kinetics in solution, leading to the equilibration of four conformational isomers for each compound. Ensemble spectroscopic measurements in solutions fail to distinguish the coexisting isomers. They reveal instead averaged absorption and emission bands with dependence of the latter on the excitation wavelength. Using high-throughput single-molecule spectroscopy, two main populations of single molecules with distinct spectral centroids are observed for each compound on glass substrates. Computational analyses suggest the two populations of molecules to be conformational isomers with antiperiplanar and periplanar arrangements of the BODIPY chromophores about its [C-C] bond to the olefinic bridge. Thus, statistical analysis of multiple single-molecule emission spectra can discriminate stereoisomers that would otherwise be impossible to distinguish by ensemble measurements alone.

Novel Curcumin Inspired Bis-Chalcone Promotes Endoplasmic Reticulum Stress and Glioblastoma Neurosphere Cell Death.

Glioblastoma (GBM) has a dismal prognosis and successful elimination of GBM stem cells (GSCs) is a high-priority as these cells are responsible for tumor regrowth following therapy and ultimately patient relapse. Natural products and their derivatives continue to be a source for the development of effective anticancer drugs and have been shown to effectively target pathways necessary for cancer stem cell self-renewal and proliferation. We generated a series of curcumin inspired bis-chalcones and examined their effect in multiple patient-derived GSC lines. Of the 19 compounds synthesized, four analogs robustly induced GSC death in six separate GSC lines, with a half maximal inhibitory concentration (IC50) ranging from 2.7⁻5.8 µM and significantly reduced GSC neurosphere formation at sub-cytotoxic levels. Structural analysis indicated that the presence of a methoxy group at position 3 of the lateral phenylic appendages was important for activity. Pathway and drug connectivity analysis of gene expression changes in response to treatment with the most active bis-chalcone 4j (the 3,4,5 trimethoxy substituted analog) suggested that the mechanism of action was the induction of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) mediated cell death. This was confirmed by Western blot analysis in which 4j induced robust increases in CHOP, p-jun and caspase 12. The UPR is believed to play a significant role in GBM pathogenesis and resistance to therapy and as such represents a promising therapeutic target.

Fluorescence activation with switchable oxazines.

The identification of operating principles to activate fluorescence under the influence of external stimulations is essential to enable the implementation of imaging strategies requiring the spatiotemporal control of emission. In this context, our laboratories designed mechanisms to switch fluorescence with either light or pH based on the unique photochemical and photophysical properties of either photoresponsive or halochromic oxazines respectively. These heterocycles can be connected covalently to fluorescent chromophores and opened with either light or pH to impose a significant bathochromic shift on the main absorption of the emissive appendage. Such a spectral change allows the selective excitation of the resulting species to activate bright fluorescence with infinite contrast and spatiotemporal control. Indeed, these mechanisms for fluorescence activation enable the acquisition of images with subdiffraction resolution, the selective signaling of cancer cells and the monitoring of translocating species in real time. Thus, our structural designs for fluorescence switching under external control can evolve into invaluable probes for the implementation of bioimaging strategies that would be impossible to perform with conventional fluorophores.

A Photoactivatable Far-Red/Near-Infrared BODIPY To Monitor Cellular Dynamics in Vivo.

A mechanism to photoactivate far-red/near-infrared fluorescence with infinite contrast and under mild visible illumination was designed around the photophysical properties of borondipyrromethene (BODIPY) dyes and the photochemical behavior of oxazine heterocycles. Specifically, the photoinduced and irreversible cleavage of an oxazine ring with a laser line at 405 nm extends the electronic conjugation of a BODIPY chromophore over a 3 H-indole auxochrome with a 2-(4-methoxyphenyl)ethenyl substituent in position 5. This structural transformation shifts bathochromically the main absorption band of the BODIPY component to allow the selective excitation of the photochemical product with a laser line of 633 nm and produce fluorescence between 600 and 850 nm. This combination of activation, excitation, and emission wavelengths permits the visualization of the cellular blastoderm of developing Drosophila melanogaster embryos with optimal contrast and essentially no autofluorescence from the biological specimen. Furthermore, the sequential acquisition of images, after the photoactivation event, enables the tracking of individual cells within the embryos in real time. Thus, our structural design and operating principles for the photoactivation of far-red/near-infrared fluorescence can evolve into invaluable probes to monitor cellular dynamics in vivo.

Semiconductor Quantum Dots with Photoresponsive Ligands.

Photochromic or photocaged ligands can be anchored to the outer shell of semiconductor quantum dots in order to control the photophysical properties of these inorganic nanocrystals with optical stimulations. One of the two interconvertible states of the photoresponsive ligands can be designed to accept either an electron or energy from the excited quantum dots and quench their luminescence. Under these conditions, the reversible transformations of photochromic ligands or the irreversible cleavage of photocaged counterparts translates into the possibility to switch luminescence with external control. As an alternative to regulating the photophysics of a quantum dot via the photochemistry of its ligands, the photochemistry of the latter can be controlled by relying on the photophysics of the former. The transfer of excitation energy from a quantum dot to a photocaged ligand populates the excited state of the species adsorbed on the nanocrystal to induce a photochemical reaction. This mechanism, in conjunction with the large two-photon absorption cross section of quantum dots, can be exploited to release nitric oxide or to generate singlet oxygen under near-infrared irradiation. Thus, the combination of semiconductor quantum dots and photoresponsive ligands offers the opportunity to assemble nanostructured constructs with specific functions on the basis of electron or energy transfer processes. The photoswitchable luminescence and ability to photoinduce the release of reactive chemicals, associated with the resulting systems, can be particularly valuable in biomedical research and can, ultimately, lead to the realization of imaging probes for diagnostic applications as well as to therapeutic agents for the treatment of cancer.