Assessment of neurotoxic effects of tri-cresyl phosphates (TCPs) and cresyl saligenin phosphate (CBDP) using a combination of in vitro techniques.

Environmental exposures to tri-cresyl phosphates (TCPs) and the possible formation of toxic metabolites (e.g. cresyl saligenin phosphate; CBDP) may cause a variety of neurotoxic effects in humans. As reported for other organophosphorus compounds (OPs), the inhibition of acetylcholine esterase (AChE) has also been proposed as the underlying mechanism for TCP neurotoxicity. The ortho-isomer, ToCP and its metabolite CBDP are also known to affect neuropathy target esterase (NTE) leading to organophosphate-induced delayed neuropathy (OPIDN). Recently, in vitro testing has led to the identification of other molecular targets and alternative mechanisms of ToCP toxicity. The metabolite CBDP and other isomers, as well as commercial mixtures have not been tested for such additional modes of actions. Accordingly, the present study investigates alterations of neurobiological correlates of central nervous processes using different in vitro techniques. The three symmetric TCP isomers - ToCP, TpCP, and TmCP - that contain a methyl group at the ortho-, para-, or meta-position of the aromatic ring system, respectively, together with a commercial TCP mixture, and CBDP were all tested using concentrations not exceeding their cytotoxic concentrations. Isolated cortical neurons were kept in culture for 6days followed by 24h incubation with different concentrations of the test compounds. Thus, all endpoints were assessed after 7days in vitro (DIV 7), at which time cell viability, neurite microstructure, and the function of glutamate receptors and voltage-gated calcium cannels (VGCC) were measured. While the cytotoxic potential of the TCP isomers and their mixture were comparable (IC50≥80µM), CBDP was more cytotoxic (IC50: 15µM) to primary cortical neurons. In contrast, CBDP (up to 10µM) did not compromise the microstructure of neurites. Ten µM of ToCP significantly reduced the size and complexity of neurite networks, but neither TmCP and TpCP nor the mixture affected this second endpoint of neurotoxicity assessment. TCPs and their mixture significantly reduced the Ca2+ influx in response to glutamate and KCl stimulation in concentrations of 10µM. Only ToCP showed a specific effect on glutamate receptors with 100nM reducing the evoked Ca2+ influx. The effects of CBDP on the provoked Ca2+ influx were much weaker than those observed for TCPs. These results confirmed that ToCP has a unique mode of action on glutamate receptors that are not observed with the metabolite CBDP and the other symmetric TCP isomers. In addition, the TmCP isomer seems to have the lowest potency with respect to inducing neurotoxic effects. CBDP did not affect the neurospecific endpoints investigated in this study. Therefore, the specific affinity of CBDP for NTE and the reported general cytotoxicity might be the most relevant modes of action of this toxic metabolite in the context of ToCP-induced neurotoxicity, including OPIDN.

Correction: Micropatterning neuronal networks.

Correction for 'Micropatterning neuronal networks' by Heike Hardelauf, et al., Analyst, 2014, 139, 3256-3264.

The involvement of TRP channels in sensory irritation: a mechanistic approach toward a better understanding of the biological effects of local irritants.

Peripheral nerves innervating the mucosae of the nose, mouth, and throat protect the organism against chemical hazards. Upon their stimulation, characteristic perceptions (e.g., stinging and burning) and various reflexes are triggered (e.g., sneezing and cough). The potency of a chemical to cause sensory irritation can be estimated by a mouse bioassay assessing the concentration-dependent decrease in the respiratory rate (50 % decrease: RD50). The involvement of the N. trigeminus and its sensory neurons in the irritant-induced decrease in respiratory rates are not well understood to date. In calcium imaging experiments, we tested which of eight different irritants (RD50 5-730 ppm) could induce responses in primary mouse trigeminal ganglion neurons. The tested irritants acetophenone, 2-ethylhexanol, hexyl isocyanate, isophorone, and trimethylcyclohexanol stimulated responses in trigeminal neurons. Most of these responses depended on functional TRPA1 or TRPV1 channels. For crotyl alcohol, 3-methyl-1-butanol, and sodium metabisulfite, no activation could be observed. 2-ethylhexanol can activate both TRPA1 and TRPV1, and at low contractions (100 µM) G protein-coupled receptors (GPCRs) seem to be involved. GPCRs might also be involved in the mediation of the responses to trimethylcyclohexanol. By using neurobiological tools, we showed that sensory irritation in vivo could be based on the direct activation of TRP channels but also on yet unknown interactions with GPCRs present in trigeminal neurons. Our results showed that the potency suggested by the RD50 values was not reflected by direct nerve-compound interaction.

RNA-Seq Analysis of Human Trigeminal and Dorsal Root Ganglia with a Focus on Chemoreceptors.

The chemosensory capacity of the somatosensory system relies on the appropriate expression of chemoreceptors, which detect chemical stimuli and transduce sensory information into cellular signals. Knowledge of the complete repertoire of the chemoreceptors expressed in human sensory ganglia is lacking. This study employed the next-generation sequencing technique (RNA-Seq) to conduct the first expression analysis of human trigeminal ganglia (TG) and dorsal root ganglia (DRG). We analyzed the data with a focus on G-protein coupled receptors (GPCRs) and ion channels, which are (potentially) involved in chemosensation by somatosensory neurons in the human TG and DRG. For years, transient receptor potential (TRP) channels have been considered the main group of receptors for chemosensation in the trigeminal system. Interestingly, we could show that sensory ganglia also express a panel of different olfactory receptors (ORs) with putative chemosensory function. To characterize OR expression in more detail, we performed microarray, semi-quantitative RT-PCR experiments, and immunohistochemical staining. Additionally, we analyzed the expression data to identify further known or putative classes of chemoreceptors in the human TG and DRG. Our results give an overview of the major classes of chemoreceptors expressed in the human TG and DRG and provide the basis for a broader understanding of the reception of chemical cues.

Impairment of glutamate signaling in mouse central nervous system neurons in vitro by tri-ortho-cresyl phosphate at noncytotoxic concentrations.

Occupational and environmental exposure to tri-cresyl phosphates (TCPs) may cause various types of neurotoxicity. Among the TCP isomers, tri-ortho-cresyl phosphate is a well-studied organophosphate (OP) known to cause OP-induced delayed neuropathy (OPIDN). Clinically, OPIDN is characterized by limb paralysis caused by the inhibition of neuropathy target esterase. Like other OPs, TOCP may also trigger acute toxicity by yet unknown mechanisms. Neurotoxic effects of TCPs, including TOCP, on central nervous system functions have not been studied in depth, and such non-OPIDN mechanisms might be related to the aerotoxic syndrome. To identify alternative mechanisms of TOCP neurotoxicity, we conducted an in vitro study using primary cortical neurons isolated from mouse embryos (E 16.5). After 24 h or 6 days in vitro (DIV), cell cultures were treated with different TOCP concentrations for 24 h. On DIV 2 and 7, we investigated three different endpoints--general cytotoxicity, neurite outgrowth, and glutamatergic signaling. At both time points, the EC50 for TOCP-induced cell death was 90 µM, however, neurite outgrowth was already significantly affected at TOCP concentrations of 10 µM. The number of cells responding to glutamate, as well as the corresponding mean response amplitudes were reduced with TOCP concentrations as low as 100 nM. For the first time, functional neurotoxicity is observed with very low TOCP concentrations, and in the absence of structural damages. Our proposed mechanism is that TOCP exposure may lead to cognitive deficits relevant in aerotoxic syndrome by inhibiting the signaling of glutamate, the most abundant excitatory neurotransmitter in the brain.

Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons.

BACKGROUND: Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes. RESULTS: Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves. CONCLUSIONS: We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission.

Micropatterning neuronal networks.

Spatially organised neuronal networks have wide reaching applications, including fundamental research, toxicology testing, pharmaceutical screening and the realisation of neuronal implant interfaces. Despite the large number of methods catalogued in the literature there remains the need to identify a method that delivers high pattern compliance, long-term stability and is widely accessible to neuroscientists. In this comparative study, aminated (polylysine/polyornithine and aminosilanes) and cytophobic (poly(ethylene glycol) (PEG) and methylated) material contrasts were evaluated. Backfilling plasma stencilled PEGylated substrates with polylysine does not produce good material contrasts, whereas polylysine patterned on methylated substrates becomes mobilised by agents in the cell culture media which results in rapid pattern decay. Aminosilanes, polylysine substitutes, are prone to hydrolysis and the chemistries prove challenging to master. Instead, the stable coupling between polylysine and PLL-g-PEG can be exploited: Microcontact printing polylysine onto a PLL-g-PEG coated glass substrate provides a simple means to produce microstructured networks of primary neurons that have superior pattern compliance during long term (>1 month) culture.

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.

Comprehensive RNA-Seq expression analysis of sensory ganglia with a focus on ion channels and GPCRs in Trigeminal ganglia.

The specific functions of sensory systems depend on the tissue-specific expression of genes that code for molecular sensor proteins that are necessary for stimulus detection and membrane signaling. Using the Next Generation Sequencing technique (RNA-Seq), we analyzed the complete transcriptome of the trigeminal ganglia (TG) and dorsal root ganglia (DRG) of adult mice. Focusing on genes with an expression level higher than 1 FPKM (fragments per kilobase of transcript per million mapped reads), we detected the expression of 12984 genes in the TG and 13195 in the DRG. To analyze the specific gene expression patterns of the peripheral neuronal tissues, we compared their gene expression profiles with that of the liver, brain, olfactory epithelium, and skeletal muscle. The transcriptome data of the TG and DRG were scanned for virtually all known G-protein-coupled receptors (GPCRs) as well as for ion channels. The expression profile was ranked with regard to the level and specificity for the TG. In total, we detected 106 non-olfactory GPCRs and 33 ion channels that had not been previously described as expressed in the TG. To validate the RNA-Seq data, in situ hybridization experiments were performed for several of the newly detected transcripts. To identify differences in expression profiles between the sensory ganglia, the RNA-Seq data of the TG and DRG were compared. Among the differentially expressed genes (> 1 FPKM), 65 and 117 were expressed at least 10-fold higher in the TG and DRG, respectively. Our transcriptome analysis allows a comprehensive overview of all ion channels and G protein-coupled receptors that are expressed in trigeminal ganglia and provides additional approaches for the investigation of trigeminal sensing as well as for the physiological and pathophysiological mechanisms of pain.

Transient receptor potential channels encode volatile chemicals sensed by rat trigeminal ganglion neurons.

Primary sensory afferents of the dorsal root and trigeminal ganglia constantly transmit sensory information depicting the individual's physical and chemical environment to higher brain regions. Beyond the typical trigeminal stimuli (e.g. irritants), environmental stimuli comprise a plethora of volatile chemicals with olfactory components (odorants). In spite of a complete loss of their sense of smell, anosmic patients may retain the ability to roughly discriminate between different volatile compounds. While the detailed mechanisms remain elusive, sensory structures belonging to the trigeminal system seem to be responsible for this phenomenon. In order to gain a better understanding of the mechanisms underlying the activation of the trigeminal system by volatile chemicals, we investigated odorant-induced membrane potential changes in cultured rat trigeminal neurons induced by the odorants vanillin, heliotropyl acetone, helional, and geraniol. We observed the dose-dependent depolarization of trigeminal neurons upon application of these substances occurring in a stimulus-specific manner and could show that distinct neuronal populations respond to different odorants. Using specific antagonists, we found evidence that TRPA1, TRPM8, and/or TRPV1 contribute to the activation. In order to further test this hypothesis, we used recombinantly expressed rat and human variants of these channels to investigate whether they are indeed activated by the odorants tested. We additionally found that the odorants dose-dependently inhibit two-pore potassium channels TASK1 and TASK3 heterologously expressed In Xenopus laevis oocytes. We suggest that the capability of various odorants to activate different TRP channels and to inhibit potassium channels causes neuronal depolarization and activation of distinct subpopulations of trigeminal sensory neurons, forming the basis for a specific representation of volatile chemicals in the trigeminal ganglia.

Trigeminal ganglion neurons of mice show intracellular chloride accumulation and chloride-dependent amplification of capsaicin-induced responses.

Intracellular Cl(-) concentrations ([Cl(-)](i)) of sensory neurons regulate signal transmission and signal amplification. In dorsal root ganglion (DRG) and olfactory sensory neurons (OSNs), Cl(-) is accumulated by the Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1), resulting in a [Cl(-)](i) above electrochemical equilibrium and a depolarizing Cl(-) efflux upon Cl(-) channel opening. Here, we investigate the [Cl(-)](i) and function of Cl(-) in primary sensory neurons of trigeminal ganglia (TG) of wild type (WT) and NKCC1(-/-) mice using pharmacological and imaging approaches, patch-clamping, as well as behavioral testing. The [Cl(-)](i) of WT TG neurons indicated active NKCC1-dependent Cl(-) accumulation. Gamma-aminobutyric acid (GABA)(A) receptor activation induced a reduction of [Cl(-)](i) as well as Ca(2+) transients in a corresponding fraction of TG neurons. Ca(2+) transients were sensitive to inhibition of NKCC1 and voltage-gated Ca(2+) channels (VGCCs). Ca(2+) responses induced by capsaicin, a prototypical stimulus of transient receptor potential vanilloid subfamily member-1 (TRPV1) were diminished in NKCC1(-/-) TG neurons, but elevated under conditions of a lowered [Cl(-)](o) suggesting a Cl(-)-dependent amplification of capsaicin-induced responses. Using next generation sequencing (NGS), we found expression of different Ca(2+)-activated Cl(-) channels (CaCCs) in TGs of mice. Pharmacological inhibition of CaCCs reduced the amplitude of capsaicin-induced responses of TG neurons in Ca(2+) imaging and electrophysiological recordings. In a behavioral paradigm, NKCC1(-/-) mice showed less avoidance of the aversive stimulus capsaicin. In summary, our results strongly argue for a Ca(2+)-activated Cl(-)-dependent signal amplification mechanism in TG neurons that requires intracellular Cl(-) accumulation by NKCC1 and the activation of CaCCs.

Anterograde transsynaptic tracing in the murine somatosensory system using Pseudorabies virus (PrV): a "live-cell"-tracing tool for analysis of identified neurons in vitro.

The Pseudorabies virus (PrV) strain Bartha is widely used as a tool for retrograde transneuronal tracing in mammals. Traced neurons can be identified in cell culture allowing the analysis of their physiological features ("live-cell"-tracing). Compared to PrV-Bartha, PrV-Kaplan is known for higher virulence and transsynaptic spread in both retrograde and anterograde direction. Herein we assess the authors assess PrV-Kaplan for transsynaptic anterograde "live-cell"-tracing. Following intranasal application in mice, labelled trigeminal and brainstem neurons could be identified in vitro. Detailed electrophysiological analysis indicated that viral infection did not affect neuronal properties, making PrV-Kaplan eligible for functional analysis of identified neurons within somatosensory systems.