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.

Human vs. Mouse Nociceptors - Similarities and Differences.

The somatosensory system allows us to detect a diverse range of physical and chemical stimuli including noxious ones, which can initiate protective reflexes to prevent tissue damage. However, the sensation of pain can - under pathological circumstances - outlive its usefulness and perpetrate ongoing suffering. Rodent model systems have been tremendously useful to help understand basic mechanisms of pain perception. Unfortunately, the translation of this knowledge into novel therapies has been challenging. We have investigated similarities and differences of human and mouse peptidergic (TRKA expressing) nociceptors using dual-color fluorescence in situ hybridization of dorsal root ganglia. By comparing the transcripts of a selected group of well-established nociceptive markers, we observed significant differences for some of them. We found co-expression of Trpv1, a key player for sensitization and inflammatory pain, with TrkA in a larger population in humans compared to mice. Similar results could be obtained for Nav1.8 and Nav1.9, two voltage gated sodium channels implicated in pathological forms of pain. Additionally, co-expression of Ret and TrkA was also found to be more abundant in human neurons. Moreover, the neurofilament heavy polypeptide was detected in all human sensory DRG neurons compared to a more selective expression pattern observed in rodents. To our knowledge, this is the first time that such detailed comparative analysis has been performed and we believe that our findings will direct future experimentation geared to understand the difficulties we face in translating findings from rodent models to humans.

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

BACKGROUND: Protein 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. METHODS: Immunoblotting 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. RESULTS: Binge 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. CONCLUSIONS: Taken 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.

PIEZO2 is required for mechanotransduction in human stem cell-derived touch receptors.

Human sensory neurons are inaccessible for functional examination, and thus little is known about the mechanisms mediating touch sensation in humans. Here we demonstrate that the mechanosensitivity of human embryonic stem (hES) cell-derived touch receptors depends on PIEZO2. To recapitulate sensory neuron development in vitro, we established a multistep differentiation protocol and generated sensory neurons via the intermediate production of neural crest cells derived from hES cells or human induced pluripotent stem (hiPS) cells. The generated neurons express a distinct set of touch receptor-specific genes and convert mechanical stimuli into electrical signals, their most salient characteristic in vivo. Strikingly, mechanosensitivity is lost after CRISPR/Cas9-mediated PIEZO2 gene deletion. Our work establishes a model system that resembles human touch receptors, which may facilitate mechanistic analysis of other sensory subtypes and provide insight into developmental programs underlying sensory neuron diversity.

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.