Astringency is a trigeminal sensation that involves the activation of G protein-coupled signaling by phenolic compounds.

Astringency is an everyday sensory experience best described as a dry mouthfeel typically elicited by phenol-rich alimentary products like tea and wine. The neural correlates and cellular mechanisms of astringency perception are still not well understood. We explored taste and astringency perception in human subjects to study the contribution of the taste as well as of the trigeminal sensory system to astringency perception. Subjects with either a lesion or lidocaine anesthesia of the Chorda tympani taste nerve showed no impairment of astringency perception. Only anesthesia of both the lingual taste and trigeminal innervation by inferior alveolar nerve block led to a loss of astringency perception. In an in vitro model of trigeminal ganglion neurons of mice, we studied the cellular mechanisms of astringency perception. Primary mouse trigeminal ganglion neurons showed robust responses to 8 out of 19 monomeric phenolic astringent compounds and 8 polymeric red wine polyphenols in Ca(2+) imaging experiments. The activating substances shared one or several galloyl moieties, whereas substances lacking the moiety did not or only weakly stimulate responses. The responses depended on Ca(2+) influx and voltage-gated Ca(2+) channels, but not on transient receptor potential channels. Responses to the phenolic compound epigallocatechin gallate as well as to a polymeric red wine polyphenol were inhibited by the Gαs inactivator suramin, the adenylate cyclase inhibitor SQ, and the cyclic nucleotide-gated channel inhibitor l-cis-diltiazem and displayed sensitivity to blockers of Ca(2+)-activated Cl(-) channels.

KIBRA (KIdney/BRAin protein) regulates learning and memory and stabilizes Protein kinase Mζ.

The WWC1 gene has been genetically associated with human episodic memory performance, and its product KIdney/BRAin protein (KIBRA) has been shown to interact with the atypical protein kinase protein kinase M ζ (PKMζ). Although recently challenged, PKMζ remains a candidate postsynaptic regulator of memory maintenance. Here, we show that PKMζ is subject to rapid proteasomal degradation and that KIBRA is both necessary and sufficient to counteract this process, thus stabilizing the kinase and maintaining its function for a prolonged time. We define the binding sequence on KIBRA, a short amino acid motif near the C-terminus. Both hippocampal knock-down of KIBRA in rats and KIBRA knock-out in mice result in decreased learning and memory performance in spatial memory tasks supporting the notion that KIBRA is a player in episodic memory. Interestingly, decreased memory performance is accompanied by decreased PKMζ protein levels. We speculate that the stabilization of synaptic PKMζ protein levels by KIBRA may be one mechanism by which KIBRA acts in memory maintenance. KIBRA/WWC1 has been genetically associated with human episodic memory. KIBRA has been shown to be post-synaptically localized, but its function remained obscure. Here, we show that KIBRA shields PKMζ, a kinase previously linked to memory maintenance, from proteasomal degradation via direct interaction. KIBRA levels in the rodent hippocampus correlate closely both to spatial memory performance in rodents and to PKMζ levels. Our findings support a role for KIBRA in memory, and unveil a novel function for this protein.

Camphor modulates TRPV3 cation channels activity by interacting with critical pore-region cysteine residues.

TRPV3 ion channels mediate thermo-transduction, nociception, inflammation and dermatitis in mammals. TRPV1-4 proteins have been shown to have conserved cysteine-residues in the pore-forming regions. These residues participate in channel activation via S-nitrosylation of channel proteins. Camphor is a commonly used ligand for TRPV3 channels. Thus the knowledge about the potential binding/interacting site(s) for camphor will help to design effective and potent analgesic compounds. In an overlap-extension PCR method, following primer-pairs were used to mutate conserved cysteine-residues in the pore-region of TRPV3 channels; GATTGAGAATcCTCCAAGGACAAAAAGGAC, TRPV3-C612S-Fw and GTCCTTGGAGgACTTCTCAATCAGTCAGTGAGG, TRPV3-C612S-Rv primers pair. And for TRPV3-C619S: GGACTCcAGTTCCTATGGCCAGC, TRPV3-C619S-Fw and GCTGGCCATAgGAACTGGAGTCC, TRPV3-C619S-Rv respectively. All cDNA constructs were confirmed by DNA-sequencing and used to make cRNAs. Oocytes expressing mTRPV3-C619S and mTRPV3-C612S mutant channels were challenged with 2-APB (1 mM), camphor (10 mM) and dihydrocarveol (10 mM) either at -40 mV or +40 mV holding potentials in voltage-clamp experiments. Responses of both mutants to 2-APB were similar to wild-type mTRPV3. Interestingly, responses to camphor were totally lost in mTRPV3-C619S mutant, while responses to dihydrocarveol remained intact. In contrast mTRPV3-C612S displayed slightly altered (16±2 % reduction) phenotype with respect to camphor sensitivity. It is concluded that pore-region cysteines play critical role in camphor sensitivity of TRPV3 ion channels.

A novel flow cytometry-based technique to measure adult neurogenesis in the brain.

The stimulation of neurogenesis is an exciting novel therapeutic option for diseases of the central nervous system, ranging from depression to neurodegeneration. One major bottleneck in screening approaches for neurogenesis-inducing compounds is the very demanding in vivo quantification of newborn neurons based on stereological techniques. To effectively develop compounds in this area, novel fast and reliable techniques for quantification of in vivo neurogenesis are needed. In this study, we introduce a flow cytometry-based method for quantifying newly generated neurons in the brain based on the counting of cell nuclei from dissected brain regions. Important steps involve density sedimentation of the cell nuclei, and staining for the proliferation marker bromodeoxy uridine and nuclear cell type markers such as NeuN. We demonstrate the ability of the technique to detect increased neurogenesis in the hippocampus of animals which underwent physical exercise and received fluoxetine treatment.

Characterization of selective TRPM8 ligands and their structure activity response (S.A.R) relationship.

PURPOSE: Transient receptor potential melastatin-8 (TRPM8) is an ion channel expressed extensively in sensory nerves, human prostate and overexpressed in a variety of cancers including prostate, breast, lung, colon and skin melanomas. It is activated by innoxious cooling and chemical stimuli. TRPM8 activation by cooling or chemical agonists is reported to induce profound analgesia in neuropathic pain conditions. Known TRPM8 agonists like menthol and icilin cross-activate other thermo-TRP channels like TRPV3 and TRPA1 and mutually inhibit TRPM8. This limits the usefulness of menthol and icilin as TRPM8 ligands. Consequently, the identification of selective and potent ligands for TRPM8 is of high relevance both in basic research and for therapeutic applications. In the present investigation, a group of menthol derivates was characterized. These ligands are selective and potent agonists of TRPM8. Interestingly they do not activate other thermo-TRPs like TRPA1, TRPV1, TRPV2, TRPV3 and TRPV4. These ion channels are also nociceptors and target of many inflammatory mediators. METHODS: Investigations were performed in a recombinant system: Xenopus oocytes microinjected with cRNA of gene of interest were superfused with the test substances after initial responses of known standard agonists. Evoked currents were measured by two-electrode voltage clamp technique. RESULTS: The newly characterized ligands possess an up to six-fold higher potency (EC50 in low microM) and an up to two-fold increase in efficacy compared to the parent compound menthol. In addition, it is found that chemical derivatives of menthol like CPS-368, CPS-369, CPS-125, WS-5 and WS-12 are the most selective ligands for TRPM8. The enhanced activity and selectivity seems to be conferred by hexacyclic ring structure present in all ligands as substances like WS-23 which lack this functional group activate TRPM8 with much lower potency (EC50 in mM) and those with pentacyclcic ring structure (furanone compounds) are totally inactive. CONCLUSION: The new substances activate TRPM8 with a higher potency, efficacy and specificity than menthol and will thus be of importance for the development of pharmacological agents suitable for treatment and diagnosis of certain cancers and as analgesics. STATEMENT OF NOVELTY: The new compounds have an unmatched specificity for TRPM8 ion channels with additional display of high potency and efficacy. Thus these substances are better pharmacological tools for TRPM8 characterization then known compounds and it is suggested that these menthol-derivates may serve as model substances for the development of TRPM8 ligands.

Histamine action on vertebrate GABAA receptors: direct channel gating and potentiation of GABA responses.

Histamine is not only a crucial cytokine in the periphery but also an important neurotransmitter and neuromodulator in the brain. It is known to act on metabotropic H1-H4 receptors, but the existence of directly histamine-gated chloride channels in mammals has been suspected for many years. However, the molecular basis of such mammalian channels remained elusive, whereas in invertebrates, genes for histamine-gated channels have been already identified. In this report, we demonstrated that histamine can directly open vertebrate ion channels and identified beta subunits of GABA(A) receptors as potential candidates for histamine-gated channels. In Xenopus oocytes expressing homomultimeric beta channels, histamine evoked currents with an EC(50) of 212 microm (beta(2)) and 174 microm (beta(3)), whereas GABA is only a very weak partial agonist. We tested several known agonists and antagonists for the histamine-binding site of H1-H4 receptors and described for beta channels a unique pharmacological profile distinct from either of these receptors. In heteromultimeric channels composed of alpha(1)beta(2) or alpha(1)beta(2)gamma(2) subunits, we found that histamine is a modulator of the GABA response rather than an agonist as it potentiates GABA-evoked currents in a gamma(2) subunit-controlled manner. Despite the vast number of synthetic modulators of GABA(A) receptors widely used in medicine, which act on several distinct sites, only a few endogenous modulators have yet been identified. We show here for the first time that histamine modulates heteromultimeric GABA(A) receptors and may thus represent an endogenous ligand for an allosteric site.

Anti-ganglioside antibodies alter presynaptic release and calcium influx.

Acute motor axonal neuropathy (AMAN) variant of Guillain-Barré syndrome is often associated with IgG anti-GM1 and -GD1a antibodies. The pathophysiological basis of antibody-mediated selective motor nerve dysfunction remains unclear. We investigated the effects of IgG anti-GM1 and -GD1a monoclonal antibodies (mAbs) on neuromuscular transmission and calcium influx in hemidiaphragm preparations and in cultured neurons, respectively, to elucidate mechanisms of Ab-mediated muscle weakness. Anti-GM1 and -GD1a mAbs depressed evoked quantal release to a significant yet different extent, without affecting postsynaptic currents. At equivalent concentrations, anti-GD1b, -GT1b, or sham mAbs did not affect neuromuscular transmission. At fourfold higher concentration, an anti-GD1b mAb (specificity described in immune sensory neuropathies) induced completely reversible blockade. In neuronal cultures, anti-GM1 and -GD1a mAbs significantly reduced depolarization-induced calcium influx. In conclusion, different anti-ganglioside mAbs induce distinct effects on presynaptic transmitter release by reducing calcium influx, suggesting that this is one mechanism of antibody-mediated muscle weakness in AMAN.

Microcontact printing of proteins for neuronal cell guidance.

The growth of neurons into networks of controlled geometry is of great interest in the field of cell-based biosensors, neuroelectronic circuits, neurological implants, pharmaceutical testing as well as fundamental biological questions about neuronal interactions. The precise control of the network architecture can be achieved by defined engineering of the surface material properties: this process is called neuronal cell patterning. Different techniques can be used to produce such surface patterns. We have chosen microcontact printing (µCP), because it is a comparatively simple and universal method for patterning biomolecules.