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) cultur

From molecules to behavior

Die sensorischen Neurone des Trigeminusnervs (5. Hirnnerv) detektieren physikalische und chemische Stimuli durch freie Nervenendigungen in den Epithelien des Kopfes und Gesichts. Die vorliegende Arbeit befasst sich mit verschiedenen Aspekten der trigeminalen Wahrnehmung chemischer Stimuli auf Ebene des Verhaltens und in Einzelzellen. Unter Anwendung psychophysischer Techniken wurden Wechselwirkungen des trigeminalen mit anderen chemosensorischen Sinnen beim Menschen untersucht, insbesondere der Einfluss des Schmeckens auf die Schmerzwahrnehmung. Polyphenole aus Tee und Rotwein, die beim Verzehr ein trockenes Mundgefühl (Adstringenz) erzeugen, wurden als adäquate trigeminale Stimuli identifiziert und die Transduktionsmechanismen untersucht. Die Arbeit befasst sich auch mit der kaum bekannten Funktion des $Na^{+}-K^{+}-2Cl^{-}-Kotransporters$ 1 im trigeminalen Sinnessystem. Hierzu wurden Experimente an trigeminalen Ganglienneuronen und Verhaltenstests an gendeletierten Mäusen durchgeführt

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

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

Amplitudes of odorant-evoked in- (−100 mV) and outward (+100 mV) currents via functionally expressed rat TRPV1, TRPM8, and TRPA1.

<p>Amplitudes of odorant-evoked in- (−100 mV) and outward (+100 mV) currents via functionally expressed rat TRPV1, TRPM8, and TRPA1.</p

CPZ and HC inhibit odorant-evoked activations of cultured TG neurons.

<p><b>A:</b> Bar chart, depicting the responsiveness (%) of TG neurons to the odorants vanillin (1 mM; n = 54), HTPA (1 mM; n = 125), helional (1 mM; n = 121), and geraniol (1 mM; n = 101), as well as to cap (3.3 µM) and men (300 µM). Detailed percentages can be derived from table S1. <b>B:</b> Bar chart, depicting the responsiveness (%) of TG neurons to the odorants vanillin (1 mM; n = 87), HTPA (1 mM; n = 68), helional (1 mM; n = 68), and geraniol (1 mM; n = 69) as well as to cap (3.3 µM) and AITC (50 µM). Detailed percentages can be derived from table S1. <b>(C-E)</b> Box plot diagrams depicting the odorant-evoked membrane potential depolarization in cultured TG neurons during whole-cell CC recordings in the presence and the absence of CPZ (C), BCTC (D) or HC (E). <b>C:</b> Recordings from menthol- and capsaicin-sensitive neurons were subdivided. Depolarizations triggered by vanillin (n = 54), HTPA (n = 70), helional (n = 76), and geraniol (n = 59) in the presence and in the absence of CPZ, as well as percentage values of inhibitions can be derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077998#pone-0077998-t004" target="_blank">table 4</a>. <b>D:</b> Recordings from menthol- and capsaicin-sensitive neurons were subdivided. Depolarizations triggered by HTPA (n = 55), helional (n = 45), and geraniol (n = 42) in the presence and in the absence of BCTC as well as percentage values of inhibitions can be derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077998#pone-0077998-t004" target="_blank">table 4</a>. <b>E:</b> Recordings from AITC/cap- and cap-sensitive neurons were subdivided, respectively (original traces are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077998#pone.0077998.s002" target="_blank">fig. S2</a>). Depolarizations triggered by vanillin (n = 87), HTPA (n = 68), helional (n = 68), and geraniol (n = 69) in the presence and in the absence of HC can be derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077998#pone-0077998-t004" target="_blank">table 4</a>.</p

Different odorants activate cultured TG neurons.

<p><b>A:</b>Box plot diagram depicting membrane potential changes in TG neurons challenged with PEE (n = 31), sandalore (n = 31), sandranol (n = 31), javanol (n = 31), vanillin (n = 68), HTPA (n = 96), helional (n = 129) and geraniol (n = 108). Significant depolarization was evoked by vanillin, HTPA, helional, and geraniol. Amplitudes can be derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077998#pone-0077998-t001" target="_blank">table 1</a>. <b>B:</b> Exemplary whole-cell CC recordings of TG neurons upon stimulation with PEE, sandalore, sandranol, javanol, vanillin, HTPA, helional, and geraniol (1 mM each). RMP: PEE: −58 mV; sandalore: −62 mV; sandranol: −62 mV; javanol: −55 mV; vanillin: −60 mV; HTPA: −61 mV; helional: −58 mV; geraniol: −55 mV. <b>C,D:</b> Box plot diagrams depicting densities (pA/pF) of activated (C) and inhibited (D) currents during whole-cell VC recordings from TG neurons upon stimulation with helional (n = 33) and geraniol (n = 32). Outward currents recorded at +100 mV are depicted by upward facing bars, inward currents recorded at −100 mV are depicted by downward facing bars. Detailed values can be derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077998#pone-0077998-t002" target="_blank">table 2</a>. <b>E,F:</b> Whole-cell VC recordings from TG neurons challenged with vanillin, sandalore, helional, geraniol, cap, and ATP depicting odorant-induced activation (E) and inhibition (F) of currents. Current amplitudes at +100 (<i>black</i>) and −100 mV (<i>gray</i>) are plotted vs. time (<i>above</i>). Stimulus application is indicated by the gray bars. Colored arrows and roman numerals assign to individual voltage ramps depicted in the corresponding IV-plots (<i>below</i>).</p