Fluorescent Tools in Neuropharmacology. Special Issue of Neuropharmacology

Neuropharmacology (Elsevier) Special Issue entitled "Fluorescent Tools in Neuropharmacology" includes ten contributions from key researchers in this field. These contributions comprise reviews and orginial research articles.

Dopaminergic treatment in idiopathic restless legs syndrome: effects on subjective sleepiness

To assess frequency and characteristics of excessive daytime sleepiness (EDS) in restless legs syndrome (RLS) and the evolution of EDS under different RLS therapies.

CSTX-1, a toxin from the venom of the hunting spider Cupiennius salei, is a selective blocker of L-type calcium channels in mammalian neurons

The inhibitor cystine-knot motif identified in the structure of CSTX-1 from Cupiennius salei venom suggests that this toxin may act as a blocker of ion channels. Whole-cell patch-clamp experiments performed on cockroach neurons revealed that CSTX-1 produced a slow voltage-independent block of both mid/low- (M-LVA) and high-voltage-activated (HVA) insect Ca(v) channels. Since C. salei venom affects both insect as well as rodent species, we investigated whether Ca(v) channel currents of rat neurons are also inhibited by CSTX-1. CSTX-1 blocked rat neuronal L-type, but no other types of HVA Ca(v) channels, and failed to modulate LVA Ca(v) channel currents. Using neuroendocrine GH3 and GH4 cells, CSTX-1 produced a rapid voltage-independent block of L-type Ca(v) channel currents. The concentration-response curve was biphasic in GH4 neurons and the subnanomolar IC(50) values were at least 1000-fold lower than in GH3 cells. L-type Ca(v) channel currents of skeletal muscle myoballs and other voltage-gated ion currents of rat neurons, such as I(Na(v)) or I(K(v)) were not affected by CSTX-1. The high potency and selectivity of CSTX-1 for a subset of L-type channels in mammalian neurons may enable the toxin to be used as a molecular tool for the investigation of this family of Ca(v) channels.

Characterizing New Fluorescent Tools for Studying 5-HT3 Receptor Pharmacology

The pharmacological characterization of ligands depends upon the ability to accurately measure their binding properties. Fluorescence provides an alternative to more traditional approaches such as radioligand binding. Here we describe the binding and spectroscopic properties of eight fluorescent 5-HT3 receptor ligands. These were tested on purified receptors, expressed receptors on live cells, or in vivo. All compounds had nanomolar affinities with fluorescent properties extending from blue to near infra-red emission. A fluorescein-derivative had the highest affinity as measured by fluorescence polarization (FP; 1.14 nM), flow cytometry (FC; 3.23 nM) and radioligand binding (RB; 1.90 nM). Competition binding with unlabeled 5-HT3 receptor agonists (5-HT, mCPBG, quipazine) and antagonists (granisetron, palonosetron, tropisetron) yielded similar affinities in all three assays. When cysteine substitutions were introduced into the 5-HT3 receptor binding site the same changes in binding affinity were seen for both granisetron and the fluorescein-derivative, suggesting that they both adopt orientations that are consistent with co-crystal structures of granisetron with a homologous protein (5HTBP). As expected, in vivo live imaging in anaesthetized mice revealed staining in the abdominal cavity in intestines, but also in salivary glands. The unexpected presence of 5-HT3 receptors in mouse salivary glands was confirmed by Western blots. Overall, these results demonstrate the wide utility of our new high-affinity fluorescently-labeled 5-HT3 receptor probes, ranging from in vitro receptor pharmacology, including FC and FP ligand competition, to live imaging of 5-HT3 expressing tissues.

Novel oxazolo-oxazole derivatives of FTY720 reduce endothelial cell permeability, immune cell chemotaxis and symptoms of experimental autoimmune encephalomyelitis in mice

The immunomodulatory FTY720 (fingolimod) is presently approved for the treatment of relapsing-remitting multiple sclerosis. It is a prodrug that acts by modulating sphingosine 1-phosphate (S1P) receptor signaling. In this study, we have developed and characterized two novel oxazolo-oxazole derivatives of FTY720, ST-968 and the oxy analog ST-1071, which require no preceding activating phosphorylation, and proved to be active in intact cells and triggered S1P1 and S1P3, but not S1P2, receptor internalization as a result of receptor activation. Functionally, ST-968 and ST-1071 acted similar to FTY720 to abrogate S1P-triggered chemotaxis of mouse splenocytes, mouse T cells and human U937 cells, and reduced TNFa- and LPS-stimulated endothelial cell permeability. The compounds also reduced TNFα-induced ICAM-1 and VCAM-1 mRNA expression, but restored TNFα-mediated downregulation of PECAM-1 mRNA expression. In an in vivo setting, the application of ST-968 or ST-1071 to mice resulted in a reduction of blood lymphocytes and significantly reduced the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice comparable to FTY720 either by prophylactic or therapeutic treatment. In parallel to the reduced clinical symptoms, infiltration of immune cells in the brain was strongly reduced, and in isolated tissues of brain and spinal cord, the mRNA and protein expressions of ICAM-1 and VCAM-1, as well as of matrix metalloproteinase-9 were reduced by all compounds, whereas PECAM-1 and tissue inhibitor of metalloproteinase TIMP-1 were upregulated. In summary, the data suggest that these novel butterfly derivatives of FTY720 could have considerable implication for future therapies of multiple sclerosis and other autoimmune diseases.

The Neuroscience of Visual Hallucinations

Each year, some two million people in the United Kingdom experience visual hallucinations. Infrequent, fleeting visual hallucinations, often around sleep, are a usual feature of life. In contrast, consistent, frequent, persistent hallucinations during waking are strongly associated with clinical disorders; in particular delirium, eye disease, psychosis, and dementia. Research interest in these disorders has driven a rapid expansion in investigatory techniques, new evidence, and explanatory models. In parallel, a move to generative models of normal visual function has resolved the theoretical tension between veridical and hallucinatory perceptions. From initial fragmented areas of investigation, the field has become increasingly coherent over the last decade. Controversies and gaps remain, but for the first time the shapes of possible unifying models are becoming clear, along with the techniques for testing these. This book provides a comprehensive survey of the neuroscience of visual hallucinations and the clinical techniques for testing these. It brings together the very latest evidence from cognitive neuropsychology, neuroimaging, neuropathology, and neuropharmacology, placing this within current models of visual perception. Leading researchers from a range of clinical and basic science areas describe visual hallucinations in their historical and scientific context, combining introductory information with up-to-date discoveries. They discuss results from the main investigatory techniques applied in a range of clinical disorders. The final section outlines future research directions investigating the potential for new understandings of veridical and hallucinatory perceptions, and for treatments of problematic hallucinations. Fully comprehensive, this is an essential reference for clinicians in the fields of the psychology and psychiatry of hallucinations, as well as for researchers in departments, research institutes and libraries. It has strong foundations in neuroscience, cognitive science, optometry, psychiatry, psychology, clinical medicine, and philosophy. With its lucid explanation and many illustrations, it is a clear resource for educators and advanced undergraduate and graduate students.

Localization and production of peptide endocannabinoids in the rodent CNS and adrenal medulla.

The endocannabinoid system (ECS) comprises the cannabinoid receptors CB1 and CB2 and their endogenous arachidonic acid-derived agonists 2-arachidonoyl glycerol and anandamide, which play important neuromodulatory roles. Recently, a novel class of negative allosteric CB1 receptor peptide ligands, hemopressin-like peptides derived from alpha hemoglobin, has been described, with yet unknown origin and function in the CNS. Using monoclonal antibodies we now identified the localization of RVD-hemopressin (pepcan-12) and N-terminally extended peptide endocannabinoids (pepcans) in the CNS and determined their neuronal origin. Immunohistochemical analyses in rodents revealed distinctive and specific staining in major groups of noradrenergic neurons, including the locus coeruleus (LC), A1, A5 and A7 neurons, which appear to be major sites of production/release in the CNS. No staining was detected in dopaminergic neurons. Peptidergic axons were seen throughout the brain (notably hippocampus and cerebral cortex) and spinal cord, indicative of anterograde axonal transport of pepcans. Intriguingly, the chromaffin cells in the adrenal medulla were also strongly stained for pepcans. We found specific co-expression of pepcans with galanin, both in the LC and adrenal gland. Using LC-MS/MS, pepcan-12 was only detected in non-perfused brain (∼40 pmol/g), suggesting that in the CNS it is secreted and present in extracellular compartments. In adrenal glands, significantly more pepcan-12 (400-700 pmol/g) was measured in both non-perfused and perfused tissue. Thus, chromaffin cells may be a major production site of pepcan-12 found in blood. These data uncover important areas of peptide endocannabinoid occurrence with exclusive noradrenergic immunohistochemical staining, opening new doors to investigate their potential physiological function in the ECS. This article is part of a Special Issue entitled 'Fluorescent Neuro-Ligands'.

A review of fluorescent ligands for studying 5-HT3 receptors

The use of fluorescence is a valuable and increasingly accessible means of probing the pharmacology and physiology of cells and their receptors. To date, the use of fluorescence-based methods for 5-HT3 receptor research has been quite limited and, although a variety of approaches have been described, these are broadly distributed throughout the literature. In this review we condense these findings into a single, accessible source of reference with the hope of promoting the use of these valuable molecular probes.

Tracking individual membrane proteins and their biochemistry: The power of direct observation

The advent of single molecule fluorescence microscopy has allowed experimental molecular biophysics and biochemistry to transcend traditional ensemble measurements, where the behavior of individual proteins could not be precisely sampled. The recent explosion in popularity of new super-resolution and super-localization techniques coupled with technical advances in optical designs and fast highly sensitive cameras with single photon sensitivity and millisecond time resolution have made it possible to track key motions, reactions, and interactions of individual proteins with high temporal resolution and spatial resolution well beyond the diffraction limit. Within the purview of membrane proteins and ligand gated ion channels (LGICs), these outstanding advances in single molecule microscopy allow for the direct observation of discrete biochemical states and their fluctuation dynamics. Such observations are fundamentally important for understanding molecular-level mechanisms governing these systems. Examples reviewed here include the effects of allostery on the stoichiometry of ligand binding in the presence of fluorescent ligands; the observation of subdomain partitioning of membrane proteins due to microenvironment effects; and the use of single particle tracking experiments to elucidate characteristics of membrane protein diffusion and the direct measurement of thermodynamic properties, which govern the free energy landscape of protein dimerization. The review of such characteristic topics represents a snapshot of efforts to push the boundaries of fluorescence microscopy of membrane proteins to the absolute limit.

Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ- subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABAA receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ- site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the β subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs.

Functional sites involved in modulation of the GABAA receptor channel by the intravenous anesthetics propofol, etomidate and pentobarbital.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs. Among the many modulatory compounds are also the intravenous anesthetics propofol and etomidate, and barbiturates. The mechanism of receptor modulation by these compounds is of mayor relevance. The site of action of these compounds has been located to subunit interfaces in the intra-membrane region of the receptor. In α1β2γ2 GABAA receptors there are five such interfaces, two β+/α- and one each of α+/β-, α+/γ- and γ+/β- subunit interfaces. We have used reporter mutations located in the second trans-membrane region in different subunits to probe the effects of changes at these subunit interfaces on modulation by propofol, etomidate and pentobarbital. We provide evidence for the fact that each of these compounds either modulates through a different set of subunit interfaces or through the same set of subunit interfaces to a different degree. As a GABAA receptor pentamer harbors two β+/α- subunit interfaces, we used concatenated receptors to dissect the contribution of individual interfaces and show that only one of these interfaces is important for receptor modulation by etomidate.

Sphingosine kinase 2 deficient mice exhibit reduced experimental autoimmune encephalomyelitis: Resistance to FTY720 but not ST-968 treatments

The immunomodulatory drug FTY720 is presently approved for the treatment of relapsing-remitting multiple sclerosis. It is a prodrug that requires activation by sphingosine kinase 2 (SK-2) to induce T cell homing to secondary lymphoid tissue. In this study, we have investigated the role of SK-2 in experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. We show that SK-2 deficiency reduced clinical symptoms of EAE. Furthermore, in SK-2-deficient mice, the protective effect of FTY720 on EAE was abolished, while the non-prodrug FTY720-derivative ST-968 was still fully active. Protection was paralleled by reduced numbers of T-lymphocytes in blood and a reduced blood-brain-barrier leakage. This correlated with reduced mRNA expression of ICAM-1, VCAM-1, but enhanced expression of PECAM-1. A similar regulation of permeability and of PECAM-1 was seen in primary cultures of isolated mouse brain vascular endothelial cells and in a human immortalized cell line upon SK-2 knockdown. In summary, these data demonstrated that deletion of SK-2 exerts a protective effect on the pathogenesis of EAE in C57BL/6 mice and that SK-2 is essential for the protective effect of FTY720 but not of ST-968. Thus, ST-968 is a promising novel immunomodulatory compound that may be a valuable alternative to FTY720 under conditions where SK-2 activity is limited.

The muscarinic antagonists scopolamine and atropine are competitive antagonists at 5-HT3 receptors

Scopolamine is a high affinity muscarinic antagonist that is used for the prevention of post-operative nausea and vomiting. 5-HT3 receptor antagonists are used for the same purpose and are structurally related to scopolamine. To examine whether 5-HT3 receptors are affected by scopolamine we examined the effects of this drug on the electrophysiological and ligand binding properties of 5-HT3A receptors expressed in Xenopus oocytes and HEK293 cells, respectively. 5-HT3 receptor-responses were reversibly inhibited by scopolamine with an IC50 of 2.09 μM. Competitive antagonism was shown by Schild plot (pA2 = 5.02) and by competition with the 5-HT3 receptor antagonists [3H]granisetron (Ki = 6.76 µM) and G-FL (Ki = 4.90 µM). The related molecule, atropine, similarly inhibited 5-HT evoked responses in oocytes with an IC50 of 1.74 µM, and competed with G-FL with a Ki of 7.94 µM. The reverse experiment revealed that granisetron also competitively bound to muscarinic receptors (Ki = 6.5 µM). In behavioural studies scopolamine is used to block muscarinic receptors and induce a cognitive deficit, and centrally administered concentrations can exceed the IC50 values found here. It is therefore possible that 5-HT3 receptors are also inhibited. Studies that utilise higher concentrations of scopolamine should be mindful of these potential off-target effects.