Functional profiling of neurons through cellular neuropharmacology

Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 01/2012; 109(5):1388-95. DOI: 10.1073/pnas.1118833109
Source: PubMed


We describe a functional profiling strategy to identify and characterize subtypes of neurons present in a peripheral ganglion, which should be extendable to neurons in the CNS. In this study, dissociated dorsal-root ganglion neurons from mice were exposed to various pharmacological agents (challenge compounds), while at the same time the individual responses of >100 neurons were simultaneously monitored by calcium imaging. Each challenge compound elicited responses in only a subset of dorsal-root ganglion neurons. Two general types of challenge compounds were used: agonists of receptors (ionotropic and metabotropic) that alter cytoplasmic calcium concentration (receptor-agonist challenges) and compounds that affect voltage-gated ion channels (membrane-potential challenges). Notably, among the latter are K-channel antagonists, which elicited unexpectedly diverse types of calcium responses in different cells (i.e., phenotypes). We used various challenge compounds to identify several putative neuronal subtypes on the basis of their shared and/or divergent functional, phenotypic profiles. Our results indicate that multiple receptor-agonist and membrane-potential challenges may be applied to a neuronal population to identify, characterize, and discriminate among neuronal subtypes. This experimental approach can uncover constellations of plasma membrane macromolecules that are functionally coupled to confer a specific phenotypic profile on each neuronal subtype. This experimental platform has the potential to bridge a gap between systems and molecular neuroscience with a cellular-focused neuropharmacology, ultimately leading to the identification and functional characterization of all neuronal subtypes at a given locus in the nervous system.

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    • "Such neuronal subclasses (defined by expression of a single gene) could eventually be further subdivided into specific neuronal cell types (defined by a particular physiological function and expression of a cell-specific combination of genes), by cross-correlating the functional expression of several different genes across many individual neurons. The basic strategy is to monitor functional activity of specific receptor- and ion-channel subtypes in more than 100 individual cells simultaneously from a heterogeneous cell population, as we described previously (Teichert et al., 2012a,b). Using this experimental approach, we characterized dissociated rat and mouse lumbar dorsal-root ganglion (DRG) neurons that express functional nAChRs, and identified the particular nAChR subtypes expressed in different neuronal subclasses. "
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    ABSTRACT: We investigated the functional expression of nicotinic acetylcholine receptors (nAChRs) in heterogeneous populations of dissociated rat and mouse lumbar dorsal root ganglion (DRG) neurons by calcium imaging. By this experimental approach, it is possible to investigate the functional expression of multiple receptor and ion-channel subtypes across more than 100 neuronal and glial cells simultaneously. Based on nAChR expression, DRG neurons could be divided into four subclasses: (1) neurons that express predominantly α3β4 and α6β4 nAChRs; (2) neurons that express predominantly α7 nAChRs; (3) neurons that express a combination of α3β4/α6β4 and α7 nAChRs; and (4) neurons that do not express nAChRs. In this comparative study, the same four neuronal subclasses were observed in mouse and rat DRG. However, the expression frequency differed between species: substantially more rat DRG neurons were in the first three subclasses than mouse DRG neurons, at all developmental time points tested in our study. Approximately 70-80% of rat DRG neurons expressed functional nAChRs, in contrast to only ~15-30% of mouse DRG neurons. Our study also demonstrated functional coupling between nAChRs, voltage-gated calcium channels, and mitochondrial Ca(2) (+) transport in discrete subsets of DRG neurons. In contrast to the expression of nAChRs in DRG neurons, we demonstrated that a subset of non-neuronal DRG cells expressed muscarinic acetylcholine receptors and not nAChRs. The general approach to comparative cellular neurobiology outlined in this paper has the potential to better integrate molecular and systems neuroscience by uncovering the spectrum of neuronal subclasses present in a given cell population and the functionally integrated signaling components expressed in each subclass.
    Frontiers in Cellular Neuroscience 11/2013; 7:225. DOI:10.3389/fncel.2013.00225 · 4.29 Impact Factor
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    • "DRG neurons are known to be heterogeneous, with a variety of different cell types that respond to different stimuli. Using the calcium-imaging assay, cell types can be identified by their differential responses to discrete reagents (Teichert et al., 2012a). "
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    • "Here, " K " indicates 25 mM KCl at minute 1 and then 15 mM KCl at subsequent time points (15 mM KCl was used because it made the amplification observed in the upper trace of B more evident, while the block observed in the lower trace of B remained clear). Teichert et al. PNAS | July 31, 2012 | vol. 109 | no. 31 | 12761 NEUROSCIENCE"
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    ABSTRACT: Different types of neurons diverge in function because they express their own unique set or constellation of signaling molecules, including receptors and ion channels that work in concert. We describe an approach to identify functionally divergent neurons within a large, heterogeneous neuronal population while simultaneously investigating specific isoforms of signaling molecules expressed in each. In this study we characterized two subclasses of menthol-sensitive neurons from cultures of dissociated mouse dorsal-root ganglia. Although these neurons represent a small fraction of the dorsal-root ganglia neuronal population, we were able to identify them and investigate the cell-specific constellations of ion channels and receptors functionally expressed in each subclass, using a panel of selective pharmacological tools. Differences were found in the functional expression of ATP receptors, TRPA1 channels, voltage-gated calcium-, potassium-, and sodium channels, and responses to physiologically relevant cold temperatures. Furthermore, the cell-specific responses to various stimuli could be altered through pharmacological interventions targeted to the cell-specific constellation of ion channels expressed in each menthol-sensitive subclass. In fact, the normal responses to cold temperature could be reversed in the two neuronal subclasses by the coapplication of the appropriate combination of pharmacological agents. This result suggests that the functionally integrated constellation of signaling molecules in a particular type of cell is a more appropriate target for effective pharmacological intervention than a single signaling molecule. This shift from molecular to cellular targets has important implications for basic research and drug discovery. We refer to this paradigm as "constellation pharmacology."
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