Differentiation and characterization of human stem cell-derived nociceptors and comparison to human and mouse dorsal root ganglia tissue

The capability to sense and transduce environmental as well as internal sensory stimuli, such as touch, pain or muscle tension, is a fundamental process required for cell survival and the avoidance of tissue damage of the body. Vertebrates can detect these stimuli via specialized cells in the peripheral nervous system - the somatosensory neurons. It is well known that the peripheral nervous system consists of many different types of neurons, but how they are generated and how they establish their functional abilities is at present not fully understood. Although pain sensation represents an adaptive alarm system detecting signals that are potentially harmful to the body, persistent pain is a maladaptive false alarm and nowadays clinicians have only few, if any, effective means to medicate chronic pain. Therefore, it is indispensable to get a more detailed understanding of how pain signals are transmitted and how human pain-sensitive neurons are established, given that most of the current knowledge about pain or pain sensation is based on animal studies. Although animal models provided a basis for research about causes, onset and course of pain diseases, there is more and more evidence that the translation of these findings to human patients is more challenging than expected. Therefore, the aim of this Ph.D. thesis was to establish a differentiation protocol for the generation of functional human embryonic stem cell (hESC)-derived nociceptors. I found that a transient overexpression of the bHLH transcription factor neurogenin 1 (NGN1), known to induce neurogenesis and to mediate the differentiation of nociceptive neurons in mice, was sufficient to differentiate progenitor cells of the peripheral nervous system (PNS) into primary sensory neurons with a nociceptive phenotype. Differentiated cells were analyzed and characterized by using Ca2+-imaging, immunohistochemistry, in situ hybridization, quantitative RT-PCR and electrophysiological recording techniques, confirming their nociceptor-like properties. To validate whether hESC-derived nociceptors are physiologically relevant and can reflect the in vivo equivalent, we compared them to human post-mortem DRG tissue where we found a similar marker gene profile. A comparative study that I carried out using human and mouse post-mortem DRG tissue highlighted molecular differences of murine and human sensory neurons that need to be considered when using the mouse as a model system for the development of new analgesic drugs. Furthermore, we were also interested in exploring the role of PIEZO2 in sensory neurons. Recent findings in rodents identified PIEZO2, a large transmembrane protein, as a main transducer of innocuous mechanical stimuli, and we confirmed that PIEZO2 is also required for mechanotransduction in human stem cell-derived touch receptors. However, it is so far uncertain whether PIEZO2 also plays a role in transducing noxious mechanical stimuli to trigger sensation of pain. Rapidly-adapting, mechanically-activated currents (at least those conventionally recorded when using a nanomotor-driven stimulus probe) appeared to be absent in PIEZO2-knockout (KO) nociceptors, indicating that PIEZO2 is also required for mechanotransduction in stem cell-derived nociceptors. Additionally, I also aimed to identify accessory proteins of PIEZO2 that are involved in human PIEZO2-mediated sensory mechanotransduction, by generating a PIEZO2-tagged hESC line. The outcome of this study would allow us to identify differences and similarities between human and mouse nociceptors and furthermore, to use this differentiation protocol as a basis for the generation of other distinct human nociceptive subpopulations, to finally provide a model system to study human pain and pain transduction in vitro

The Wooster Voice (Wooster, OH), 1949-12-08

Dr. T. Cuyler Young addresses the campus during the annual Wooster Day celebration. Dr. Delbert Lean will give his 40th annual reading of Charles Dickens\u27 Christmas Carol. Plans to build a darkroom for student publications are announced. Additionally, Wooster host the fall conference of the Ohio division of the National Student Association.https://openworks.wooster.edu/voice1941-1950/1204/thumbnail.jp

mGluR1 within the nucleus accumbens regulates alcohol intake in mice under limited-access conditions

Idiopathic or alcohol-induced increases in the expression and function of the Group1 metabotropic glutamate receptor subtype 1 (mGluR1) within the extended amygdala are theorized to contribute to an individual's propensity to consume excessive amounts of alcohol. In the past, the detailed study of the functional relevance of mGluR1 for alcoholism-related behaviors in animal models was hampered by the poor solubility and non-specific side effects of available inhibitors; however, the advent of the highly potent and soluble mGluR1 negative allosteric modulator JNJ-16259685 [(3,4-Dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone] has instigated a re-examination of the role for this mGluR subtype in mediating the behavioral effects of alcohol. In this regard, systemic pretreatment with JNJ-16259685 was proven effective at reducing alcohol reinforcement and motivation for the drug. mGluR1 is a Gαq/o-coupled receptor, the stimulation of which activates phospholipase C (PLC). Thus, the present study investigated potential neuroanatomical substrates and intracellular molecules involved in the ability of JNJ-16259685 to reduce alcohol intake. JNJ-16259685 (0-30 pg/side) was infused into the shell subregion of the nucleus accumbens (NAC) of C57BL/6J and Homer2 knock-out (KO) mice, either alone or in combination with the PLC inhibitor U-73122 (5.8 fg/side). Alcohol intake was then assessed under Drinking-in-the-Dark (DID) procedures. Intra-NAC JNJ-16259685 infusion dose-dependently reduced alcohol consumption by C57BL/6J mice; this effect was not additive with that produced by U-73122, nor was it present in Homer2 KO animals. These data provide novel evidence in support of a critical role for mGluR1-PLC signaling, scaffolded by Homer2, within the NAC shell, in maintaining alcohol consumption under limited access procedures. Such findings have relevance for both the pharmacotherapeutics and pharmacogenetics of risky alcohol drinking and alcoholism

Human Stem Cell-Derived TRPV1-Positive Sensory Neurons : A New Tool to Study Mechanisms of Sensitization

Somatosensation, the detection and transduction of external and internal stimuli such as temperature or mechanical force, is vital to sustaining our bodily integrity. But still, some of the mechanisms of distinct stimuli detection and transduction are not entirely understood, especially when noxious perception turns into chronic pain. Over the past decade major progress has increased our understanding in areas such as mechanotransduction or sensory neuron classification. However, it is in particular the access to human pluripotent stem cells and the possibility of generating and studying human sensory neurons that has enriched the somatosensory research field. Based on our previous work, we describe here the generation of human stem cell-derived nociceptor-like cells. We show that by varying the differentiation strategy, we can produce different nociceptive subpopulations with different responsiveness to nociceptive stimuli such as capsaicin. Functional as well as deep sequencing analysis demonstrated that one protocol in particular allowed the generation of a mechano-nociceptive sensory neuron population, homogeneously expressing TRPV1. Accordingly, we find the cells to homogenously respond to capsaicin, to become sensitized upon inflammatory stimuli, and to respond to temperature stimulation. The efficient and homogenous generation of these neurons make them an ideal translational tool to study mechanisms of sensitization, also in the context of chronic pain.Funding Agencies|German Research Foundation [SCHR1523/2-1, SFB-1158]; Intramural Program of the NIH, the National Center for Complementary and Integrative Health</p

Protein Kinase C Epsilon Activity in the Nucleus Accumbens and Central Nucleus of the Amygdala Mediates Binge Alcohol Consumption.

BackgroundProtein kinase C epsilon (PKCε) is emerging as a potential target for the development of pharmacotherapies to treat alcohol use disorders, yet little is known regarding how a history of a highly prevalent form of drinking, binge alcohol intake, influences enzyme priming or the functional relevance of kinase activity for excessive alcohol intake.MethodsImmunoblotting was employed on tissue from subregions of the nucleus accumbens (NAc) and the amygdala to examine both idiopathic and binge drinking-induced changes in constitutive PKCε priming. The functional relevance of PKCε translocation for binge drinking and determination of potential upstream signaling pathways involved were investigated using neuropharmacologic approaches within the context of two distinct binge drinking procedures, drinking in the dark and scheduled high alcohol consumption.ResultsBinge alcohol drinking elevated p(Ser729)-PKCε levels in both the NAc and the central nucleus of the amygdala (CeA). Moreover, immunoblotting studies of selectively bred and transgenic mouse lines revealed a positive correlation between the propensity to binge drink alcohol and constitutive p(Ser729)-PKCε levels in the NAc and CeA. Finally, neuropharmacologic inhibition of PKCε translocation within both regions reduced binge alcohol consumption in a manner requiring intact group 1 metabotropic glutamate receptors, Homer2, phospholipase C, and/or phosphotidylinositide-3 kinase function.ConclusionsTaken together, these data indicate that PKCε signaling in both the NAc and CeA is a major contributor to binge alcohol drinking and to the genetic propensity to consume excessive amounts of alcohol