Photoswitchable carbamazepine analogs for non-invasive neuroinhibition in vivo.

A problem of systemic pharmacotherapy is off-target activity, which causes adverse effects. Outstanding examples include neuroinhibitory medications like antiseizure drugs, which are used against epilepsy and neuropathic pain but cause systemic side effects. There is a need of drugs that inhibit nerve signals locally and on-demand without affecting other regions of the body. Photopharmacology aims to address this problem with light-activated drugs and localized illumination in the target organ. Here, we have developed photoswitchable derivatives of the widely prescribed antiseizure drug carbamazepine. For that purpose, we expanded our method of ortho azologization of tricyclic drugs to meta/para and to N-bridged diazocine. Our results validate the concept of ortho cryptoazologs (uniquely exemplified by Carbazopine-1) and bring to light Carbadiazocine (8), which can be photoswitched between 400-590 nm light (using violet LEDs and halogen lamps) and shows good drug-likeness and predicted safety. Both compounds display photoswitchable activity in vitro and in translucent zebrafish larvae. Carbadiazocine (8) also offers in vivo analgesic efficacy (mechanical and thermal stimuli) in a rat model of neuropathic pain and a simple and compelling treatment demonstration with non-invasive illumination.

Donor-Acceptor Stenhouse Adduct Displaying Reversible Photoswitching in Water and Neuronal Activity.

The interest in the photochromism and functional applications of donor-acceptor Stenhouse adducts (DASAs) soared in recent years owing to their outstanding advantages and flexible design. However, their low solubility and irreversible conversion in aqueous solutions hampered exploring DASAs for biology and medicine. It is notably unknown whether the barbiturate electron acceptor group retains the pharmacological activity of drugs such as phenobarbital, which targets γ-aminobutyric acid (GABA)-type A receptors (GABAARs) in the brain. Here, we have developed the model compound DASA-barbital based on a scaffold of red-switching second-generation DASAs, and we demonstrate that it is active in GABAARs and alters the neuronal firing rate in a physiological medium at neutral pH. DASA-barbital can also be reversibly photoswitched in acidic aqueous solutions using cyclodextrin, an approved ingredient of drug formulations. These findings clarify the path toward the biological applications of DASAs and to exploit the versatility displayed in polymers and materials science.

Optical Control of GABAA Receptors with a Fulgimide-Based Potentiator.

Optogenetic and photopharmacological tools to manipulate neuronal inhibition have limited efficacy and reversibility. We report the design, synthesis, and biological evaluation of Fulgazepam, a fulgimide derivative of benzodiazepine that behaves as a pure potentiator of ionotropic γ-aminobutyric acid receptors (GABAA Rs) and displays full and reversible photoswitching in vitro and in vivo. The compound enables high-resolution studies of GABAergic neurotransmission, and phototherapies based on localized, acute, and reversible neuroinhibition.

Azologization of serotonin 5-HT3 receptor antagonists.

The serotonin 5-hydroxytryptamine 3 receptor (5-HT3R) plays a unique role within the seven classes of the serotonin receptor family, as it represents the only ionotropic receptor, while the other six members are G protein-coupled receptors (GPCRs). The 5-HT3 receptor is related to chemo-/radiotherapy provoked emesis and dysfunction leads to neurodevelopmental disorders and psychopathologies. Since the development of the first serotonin receptor antagonist in the early 1990s, the range of highly selective and potent drugs expanded based on various chemical structures. Nevertheless, on-off-targeting of a pharmacophore's activity with high spatiotemporal resolution as provided by photopharmacology remains an unsolved challenge bearing additionally the opportunity for detailed receptor examination. In the presented work, we summarize the synthesis, photochromic properties and in vitro characterization of azobenzene-based photochromic derivatives of published 5-HT3R antagonists. Despite reported proof of principle of direct azologization, only one of the investigated derivatives showed antagonistic activity lacking isomer specificity.

Glycine receptors are involved in hippocampal neuronal damage caused by oxygen-glucose deficiency.

Glycine receptors (GlyRs) belong to the family of ligand-gated cys-loop receptors and effectuate fast inhibitory neurotransmission in central nervous system (CNS). They are involved in numerous physiological processes, such as movement, respiration, and processing of sensory information, as well as in regulation of neuronal excitability in different brain regions. GlyRs play important role in the maintenance of excitatory/inhibitory balance in the hippocampus and participate in the development of various brain pathologies. In the present study, we have examined a surface expression of GlyRs by pyramidal neurons and astrocytes in control and after 30 min of oxygen-glucose deprivation (OGD) in the organotypic culture of hippocampal slices. Our investigation has demonstrated a decrease in GlyR-positive staining associated with pyramidal neurons and relative stability of GlyRs expression at the surface of astrocytes 4 hs after OGD. These data indicate that GlyRs dysfunction may represent a significant additional factor leading to enhanced neuronal damage induced by OGD. Pharmacological modulation of GlyRs is a promising venue of research for the correction of negative consequences of oxygen-glucose deficiency.

Orthogonal Control of Neuronal Circuits and Behavior Using Photopharmacology.

Over the last decades, photopharmacology has gone far beyond its proof-of-concept stage to become a bona fide approach to study neural systems in vivo. Indeed, photopharmacological control has expanded over a wide range of endogenous targets, such as receptors, ion channels, transporters, kinases, lipids, and DNA transcription processes. In this review, we provide an overview of the recent progresses in the in vivo photopharmacological control of neuronal circuits and behavior. In particular, the use of small aquatic animals for the in vivo screening of photopharmacological compounds, the recent advances in optical modulation of complex behaviors in mice, and the development of adjacent techniques for light and drug delivery in vivo are described.

Subunit-Specific Photocontrol of Glycine Receptors by Azobenzene-Nitrazepam Photoswitcher.

Photopharmacology is a unique approach that through a combination of photochemistry methods and advanced life science techniques allows the study and control of specific biological processes, ranging from intracellular pathways to brain circuits. Recently, a first photochromic channel blocker of anion-selective GABAA receptors, the azobenzene-nitrazepam-based photochromic compound (Azo-NZ1), has been described. In the present study, using patch-clamp technique in heterologous system and in mice brain slices, site-directed mutagenesis and molecular modeling we provide evidence of the interaction of Azo-NZ1 with glycine receptors (GlyRs) and determine the molecular basis of this interaction. Glycinergic synaptic neurotransmission determines an important inhibitory drive in the vertebrate nervous system and plays a crucial role in the control of neuronal circuits in the spinal cord and brain stem. GlyRs are involved in locomotion, pain sensation, breathing, and auditory function, as well as in the development of such disorders as hyperekplexia, epilepsy, and autism. Here, we demonstrate that Azo-NZ1 blocks in a UV-dependent manner the activity of α2 GlyRs (GlyR2), while being barely active on α1 GlyRs (GlyR1). The site of Azo-NZ1 action is in the chloride-selective pore of GlyR at the 2' position of transmembrane helix 2 and amino acids forming this site determine the difference in Azo-NZ1 blocking activity between GlyR2 and GlyR1. This subunit-specific modulation is also shown on motoneurons of brainstem slices from neonatal mice that switch during development from expressing "fetal" GlyR2 to "adult" GlyR1 receptors.

Photocontrol of Endogenous Glycine Receptors In Vivo.

Glycine receptors (GlyRs) are indispensable for maintaining excitatory/inhibitory balance in neuronal circuits that control reflexes and rhythmic motor behaviors. Here we have developed Glyght, a GlyR ligand controlled with light. It is selective over other Cys-loop receptors, is active in vivo, and displays an allosteric mechanism of action. The photomanipulation of glycinergic neurotransmission opens new avenues to understanding inhibitory circuits in intact animals and to developing drug-based phototherapies.

A photoswitchable GABA receptor channel blocker.

BACKGROUND AND PURPOSE: Anion-selective Cys-loop receptors (GABA and glycine receptors) provide the main inhibitory drive in the CNS. Both types of receptor operate via chloride-selective ion channels, though with different kinetics, pharmacological profiles, and localization. Disequilibrium in their function leads to a variety of disorders, which are often treated with allosteric modulators. The few available GABA and glycine receptor channel blockers effectively suppress inhibitory currents in neurons, but their systemic administration is highly toxic. With the aim of developing an efficient light-controllable modulator of GABA receptors, we constructed azobenzene-nitrazepam (Azo-NZ1), which is composed of a nitrazepam moiety merged to an azobenzene photoisomerizable group. EXPERIMENTAL APPROACH: The experiments were carried out on cultured cells expressing Cys-loop receptors of known subunit composition and in brain slices using patch-clamp. Site-directed mutagenesis and molecular modelling approaches were applied to evaluate the mechanism of action of Azo-NZ1. KEY RESULTS: At visible light, being in trans-configuration, Azo-NZ1 blocked heteromeric α1/ß2/γ2 GABAA receptors, ρ2 GABAA (GABAC ), and α2 glycine receptors, whereas switching the compound into cis-state by UV illumination restored the activity. Azo-NZ1 successfully photomodulated GABAergic currents recorded from dentate gyrus neurons. We demonstrated that in trans-configuration, Azo-NZ1 blocks the Cl-selective ion pore of GABA receptors interacting mainly with the 2' level of the TM2 region. CONCLUSIONS AND IMPLICATIONS: Azo-NZ1 is a soluble light-driven Cl-channel blocker, which allows photo-modulation of the activity induced by anion-selective Cys-loop receptors. Azo-NZ1 is able to control GABAergic postsynaptic currents and provides new opportunities to study inhibitory neurotransmission using patterned illumination.

Light-induced regulation of ligand-gated channel activity.

The control of ligand-gated receptors with light using photochromic compounds has evolved from the first handcrafted examples to accurate, engineered receptors, whose development is supported by rational design, high-resolution protein structures, comparative pharmacology and molecular biology manipulations. Photoswitchable regulators have been designed and characterized for a large number of ligand-gated receptors in the mammalian nervous system, including nicotinic acetylcholine, glutamate and GABA receptors. They provide a well-equipped toolbox to investigate synaptic and neuronal circuits in all-optical experiments. This focused review discusses the design and properties of these photoswitches, their applications and shortcomings and future perspectives in the field. LINKED ARTICLES: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.

Developmental Changes in the Inhibition of Glycinergic Synaptic Currents by Niflumic Acid in Hypoglossal Motoneurons.

Mammalian brainstem hypoglossal motoneurones (HMs) receive powerful synaptic glycinergic inputs and are involved in a variety of motor functions, including respiration, chewing, sucking, swallowing, and phonation. During the early postnatal development, subunit composition of chloride-permeable glycine receptors (GlyRs) changes leading to a decrease of "fetal" alpha2 and elevation of "adult" alpha1 GlyR subunits. It has been recently demonstrated that niflumic acid (NFA), a member of the fenamate class of non-steroidal anti-inflammatory drugs, is an efficient subunits-specific blocker of GlyRs. At a heterologous expression of different GlyR subunits it has been shown that blocking potency of NFA is more than one order higher for alpha2 GlyRs than for receptors formed by alpha1 subunit. To reveal the action of NFA on the synaptic activity we analyzed here the effects of NFA on the glycinergic inhibitory post-synaptic currents in the HMs from mouse brainstem slices. In the whole-cell patch clamp configuration, the amplitude and the frequency of glycinergic synaptic currents from two age groups have been analyzed: "neonate" (P2-P4) and "juvenile" (P7-P12). Addition of NFA in the presence of antagonists of glutamate and GABA receptors caused a decrease in the mean amplitude and frequency of synaptic events. The degree of the inhibition induced by NFA decreased with the postnatal development, being higher on the motoneurons from "neonate" brainstem slices in comparison with the "juvenile" age group. Analysis of the pair-pulse facilitation suggests the post-synaptic origin of NFA action. These observations provide evidence on the developmental changes in the inhibition by NFA of glycinergic synaptic transmission, which reflects increase in the alpha1 and decrease in the alpha2 GlyR subunits expression in synapses to hypoglossal motoneurons during the early stages of postnatal life.

Voltage-Dependent Inhibition of Glycine Receptor Channels by Niflumic Acid.

Niflumic acid (NFA) is a member of the fenamate class of nonsteroidal anti-inflammatory drugs. This compound and its derivatives are used worldwide clinically for the relief of chronic and acute pain. NFA is also a commonly used blocker of voltage-gated chloride channels. Here we present evidence that NFA is an efficient blocker of chloride-permeable glycine receptors (GlyRs) with subunit heterogeneity of action. Using the whole-cell configuration of patch-clamp recordings and molecular modeling, we analyzed the action of NFA on homomeric α1ΔIns, α2B, α3L, and heteromeric α1ß and α2ß GlyRs expressed in CHO cells. NFA inhibited glycine-induced currents in a voltage-dependent manner and its blocking potency in α2 and α3 GlyRs was higher than that in α1 GlyR. The Woodhull analysis suggests that NFA blocks α1 and α2 GlyRs at the fractional electrical distances of 0.16 and 0.65 from the external membrane surface, respectively. Thus, NFA binding site in α1 GlyR is closer to the external part of the membrane, while in α2 GlyR it is significantly deeper in the pore. Mutation G254A at the cytoplasmic part of the α1 GlyR pore-lining TM2 helix (level 2') increased the NFA blocking potency, while incorporation of the ß subunit did not have a significant effect. The Hill plot analysis suggests that α1 and α2 GlyRs are preferably blocked by two and one NFA molecules, respectively. Molecular modeling using Monte Carlo energy minimizations provides the structural rationale for the experimental data and proposes more than one interaction site along the pore where NFA can suppress the ion permeation.

Selective potentiation of alpha 1 glycine receptors by ginkgolic acid.

Glycine receptors (GlyRs) belong to the superfamily of pentameric cys-loop receptor-operated channels and are involved in numerous physiological functions, including movement, vision, and pain. In search for compounds performing subunit-specific modulation of GlyRs we studied action of ginkgolic acid, an abundant Ginkgo biloba product. Using patch-clamp recordings, we analyzed the effects of ginkgolic acid in concentrations from 30 nM to 25 µM on α1-α3 and α1/ß, α2/ß configurations of GlyR and on GABAARs expressed in cultured CHO-K1 cells and mouse neuroblastoma (N2a) cells. Ginkgolic acid caused an increase in the amplitude of currents mediated by homomeric α1 and heteromeric α1/ß GlyRs and provoked a left-shift of the concentration-dependent curves for glycine. Even at high concentrations (10-25 µM) ginkgolic acid was not able to augment ionic currents mediated by α2, α2/ß, and α3 GlyRs, or by GABAAR consisting of α1/ß2/γ2 subunits. Mutation of three residues (T59A/A261G/A303S) in the α2 GlyR subunit to the corresponding ones from the α1 converted the action of ginkgolic acid to potentiation with a distinct decrease in EC50 for glycine, suggesting an important role for these residues in modulation by ginkgolic acid. Our results suggest that ginkgolic acid is a novel selective enhancer of α1 GlyRs.

Efficient and cost-effective generation of mature neurons from human induced pluripotent stem cells.

For years, our ability to study pathological changes in neurological diseases has been hampered by the lack of relevant models until the recent groundbreaking work from Yamanaka's group showing that it is feasible to generate induced pluripotent stem cells (iPSCs) from human somatic cells and to redirect the fate of these iPSCs into differentiated cells. In particular, much interest has focused on the ability to differentiate human iPSCs into neuronal progenitors and functional neurons for relevance to a large number of pathologies including mental retardation and behavioral or degenerative syndromes. Current differentiation protocols are time-consuming and generate limited amounts of cells, hindering use on a large scale. We describe a feeder-free method relying on the use of a chemically defined medium that overcomes the need for embryoid body formation and neuronal rosette isolation for neuronal precursors and terminally differentiated neuron production. Four days after induction, expression of markers of the neurectoderm lineage is detectable. Between 4 and 7 days, neuronal precursors can be expanded, frozen, and thawed without loss of proliferation and differentiation capacities or further differentiated. Terminal differentiation into the different subtypes of mature neurons found in the human brain were observed. At 6-35 days after induction, cells express typical voltage-gated and ionotrophic receptors for GABA, glycine, and acetylcholine. This specific and efficient single-step strategy in a chemically defined medium allows the production of mature neurons in 20-40 days with multiple applications, especially for modeling human pathologies.

Comparative ultrastructural analysis of mitochondria in the CA1 and CA3 hippocampal pyramidal cells following global ischemia in Mongolian gerbils.

Post-ischemic injury of the hippocampus unrolls at different levels and has both functional and structural implications. The deficiency in neuron energy metabolism is an initiating factor. We performed transmission electron microscopic (TEM) comparative analysis of mitochondria in excitatory spine synapses in CA1 stratum radiatum and CA3 hippocampal areas after 5 min of global cerebral ischemia in Mongolian gerbils, 4 and 7 days after reperfusion. Electron microscopy and unbiased morphometric methods were used to evaluate synaptic plasticity, and the number and size of mitochondria in synaptic terminals. We compared the morphological organization of mitochondria in presynaptic terminals between CA1 and CA3 areas in control and post-ischemic condition according to the following morphometric parameters: mitochondrial volume fraction, mitochondrial frequency in CA1 and CA3 terminals, mean number of mitochondria per presynaptic terminal, frequency of damaged mitochondria in terminals, and density of presynaptic terminals. Our ultrastructural study revealed statistically significant differences in morphometric parameters between CA1 and CA3 areas in control conditions, as well as in post-ischemic conditions. Also, we found temporal differences in measured parameters obtained 4 and 7 days after reperfusion. This study showed significant morphological differences in the organization of mitochondria in excitatory spine synapses between CA1 and CA3 areas, which corresponded with already known differences in functionality and sensitivity to the ischemic insult. Our conclusion is that revealed post-ischemic changes in mitochondrial distribution in presynaptic CA1 and CA3 terminals could be an indicator of hippocampal metabolic dysfunction and synaptic plasticity.