Functional profiling of neurons throughcellular neuropharmacologyRussell W. Teicherta,1, Nathan J. Smitha, Shrinivasan Raghuramana,b, Doju Yoshikamia, Alan R. Lightc,and Baldomero M. Oliveraa,1

aDepartment of Biology, University of Utah, Salt Lake City, UT 84112; bSchool of Chemical & Biotechnology, Shanmugha Arts, Science, Technology & ResearchAcademy (SASTRA) University, Tirumalaisamudram, Thanjavur 613401, Tamilnadu, India; and cDepartment of Anesthesiology, University of Utah School ofMedicine, Salt Lake City, UT 84132

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2009.

Contributed by Baldomero M. Olivera, November 21, 2011 (sent for review August 13, 2011)

We describe a functional profiling strategy to identify and charac-terize subtypes of neurons present in a peripheral ganglion, whichshould be extendable to neurons in the CNS. In this study, dissoci-ated dorsal-root ganglion neurons from mice were exposed tovarious pharmacological agents (challenge compounds), while atthe same time the individual responses of>100 neuronswere simul-taneously monitored by calcium imaging. Each challenge compoundelicited responses in only a subset of dorsal-root ganglion neurons.Two general types of challenge compounds were used: agonists ofreceptors (ionotropic and metabotropic) that alter cytoplasmic cal-cium concentration (receptor–agonist challenges) and compoundsthat affect voltage-gated ion channels (membrane–potential chal-lenges). Notably, among the latter are K-channel antagonists,which elicited unexpectedly diverse types of calcium responses indifferent cells (i.e., phenotypes). We used various challenge com-pounds to identify several putative neuronal subtypes on the basisof their shared and/or divergent functional, phenotypic profiles.Our results indicate that multiple receptor–agonist and mem-brane–potential challenges may be applied to a neuronal popula-tion to identify, characterize, and discriminate among neuronalsubtypes. This experimental approach can uncover constellationsof plasmamembranemacromolecules that are functionally coupledto confer a specific phenotypic profile on each neuronal subtype.This experimental platform has the potential to bridge a gap be-tween systems and molecular neuroscience with a cellular-focusedneuropharmacology, ultimately leading to the identification andfunctional characterization of all neuronal subtypes at a given locusin the nervous system.

sensory neuron | neuronal subpopulation | conotoxin | conopeptide | Fura-2

For any mechanistic investigation of nervous system function, itis essential to identify the neurons involved. However, this

identification can be extremely challenging, because at any givenlocus in the mammalian nervous system, many functionally di-verse neurons can be present that are difficult to discriminatefrom each other. This lack of cellular differentiation createsa barrier between systems and molecular neuroscience. Systemsneuroscientists characterize properties of circuits, whereas mo-lecular neuroscientists identify the signaling macromoleculesthat give neurons specific functional properties. Within in-vertebrate nervous systems, there is a precedent for identifyingdifferent types of neurons morphologically, anatomically, andphysiologically, known as the “identified neuron approach” (1).Progress in understanding the mammalian nervous system hasbeen impeded by the lack of a similar paradigm. The gap be-tween systems and molecular neuroscience in the mammaliannervous system could be narrowed if there were a straightfor-ward methodology to identify different neuronal subtypes.We define neuronal subtype as a neuronal cell with a specific

physiological function in contrast to a neuronal subpopulation orneuronal subclass, which may encompass multiple neuronalsubtypes and be identified by the use of a single structural or

functional marker. For example, dorsal-root ganglion (DRG)neurons may be subdivided into neuronal subclasses defined bystaining with fluorescently labeled isolectin B4 (IB4), which bindsto the extracellular matrix proteoglycan, versican, present on theplasma membranes of a subset of relatively small neurons.However, neither the IB4-positive nor -negative subclass is ho-mogeneous (e.g., both subclasses include capsaicin-sensitive and-resistant neurons) (2). A single marker is rarely unique to aspecific neuronal subtype (3–5), particularly at a complex ana-tomical locus, where potentially hundreds of neuronal subtypeswith different physiological roles may be present.Although the mammalian nervous system has been studied

intensively at the cellular level for decades, there is no ana-tomical locus where all of the neuronal subtypes have beenidentified. From a long tradition of anatomical studies pio-neered by Santiago Ramón y Cajal (6), neurons that have beenmost intensively investigated, such as Purkinje and pyramidalcells, are those cells easily recognized by their striking cellshapes. However, even morphologically similar pyramidal cellshave been classified into different subclasses based on differentfiring properties (7). In most regions of the nervous system, thevast majority of neurons are not easily differentiated mor-phologically, further complicating the task of parsing out aspecific neuronal subtype from the surrounding anatomicallysimilar neurons.Most efforts to differentiate neuronal subtypes broadly use

markers of mRNA or protein expression (3–5, 8–11). Only a fewmarkers can be used simultaneously, and the expression ofmRNA, or even protein, may not correlate with functional ex-pression; however, functional expression is the critical parameterfor any mechanistic or physiological study. The principle methodfor assaying neuronal function has been patch-clamp electro-physiology. However, it is severely limited by throughput; ex-periments are usually conducted on one neuron at a time.In this report, we identify different neuronal subtypes using an

experimental strategy that overcomes many of the limitations ofother methods. For this study, we applied pharmacological agents(challenge compounds) to dissociated mouse lumbar DRG neu-rons, while monitoring the responses of >100 individual neuronalcells simultaneously by calcium imaging. Within DRG, >25 sub-types of neurons are believed to be present based on differentsensory modalities. The divergent responses of individual cellsto each challenge compound served as the primary criteria for

Author contributions: R.W.T. and B.M.O. designed research; R.W.T., N.J.S., and S.R. per-formed research; R.W.T., D.Y., A.R.L., and B.M.O. analyzed data; and R.W.T., D.Y., A.R.L.,and B.M.O. wrote the paper.

The authors declare no conflict of interest.1To whom correspondence may be addressed. E-mail: russ_teichert@yahoo.com orolivera@biology.utah.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1118833109/-/DCSupplemental.

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distinguishing between neuronal subtypes. The rationale is thatdifferent neuronal subtypes express different receptors and ionchannels in their plasma membranes, which create functionaldivergence. The cell bodies of DRG neurons generally are goodsurrogates for functional protein expression in axons and nerveendings based on consistency of responses to selective pharma-cological agents obtained in cell bodies, nerve fibers, nerve end-ings, and behavioral studies in vivo with WT and KO mice (12).We have used an established technology, calcium imaging, to

profile neuronal subtypes. Although this approach is not un-precedented, typically only a few pharmacological agents havebeen used to profile neuronal subtypes in a single experiment(13–15). We show the feasibility of applying many challengecompounds in a single experiment, and we have discovered thatcertain types of challenge compounds elicited a far greaterspectrum of phenotypic responses than predicted. The successfulapplication of many challenge compounds, coupled with theunexpected diversity of response phenotypes, establishes thisexperimental strategy as a powerful approach to distinguish be-tween neuronal subtypes in a heterogeneous cell population.Using this experimental approach, we highlight a few un-ambiguous examples of neuronal subclasses in DRG cultures.Some of these neuronal subclasses probably conform to ournarrow definition of a neuronal subtype (with a shared, specificphysiological function), because each neuron within the subclassshares a common structural and functional profile (includingboth positive and negative markers) that clearly distinguishes itfrom other neuronal subclasses in the DRG cultures.

ResultsPreparation and Calcium Imaging of Dissociated Mouse Lumbar DRGNeurons. Fig. 1 shows bright-field and fluorescence images ofcultured DRG neurons loaded with Fura-2-acetoxymethyl ester

(Fura-2-AM) and imaged as described in Materials and Methods.The wide range of cell diameters observed in Fig. 1A is consistentwith previous reports (16). Also shown are ratiometric imagesacquired before and during exposure to a high concentration ofpotassium (Fig. 1 C and D).Cultured DRG neurons, similar to those neurons shown in

Fig. 1, were challenged using two types of experimental proto-cols. The first type of protocol used agonists of receptors (ion-otropic and metabotropic) previously reported to produce anincrease in cytoplasmic calcium concentration, [Ca2+]i, of DRGneurons. We refer to this experimental strategy as a receptor–agonist challenge (RA challenge). The second type of protocolused pharmacological agents targeted to voltage-gated ionchannels, which perturb the membrane potential. A membranedepolarization that activates voltage-gated Ca channels willproduce an increase in [Ca2+]i. Thus, compounds that act onvoltage-gated ion channels may attenuate or enhance the in-crease in [Ca2+]i elicited by a membrane depolarization. Werefer to this experimental strategy as a membrane–potentialchallenge (MP challenge).

RA Challenge Protocol. With ionotropic receptors (ligand-gatedion channels), RA challenge compounds directly induce the in-flux of Ca2+ through Ca2+-permeable channels [e.g., transientreceptor potential channel (TRP) V1 receptor] (17). Withmetabotropic receptors (G protein-coupled receptors), RAchallenge compounds indirectly elevate cytoplasmic Ca2+ bydownstream signaling pathways that ultimately trigger the re-lease of Ca2+ from the endoplasmic reticulum (e.g., histaminereceptors) (18–20). Six different RA challenge compounds weresequentially applied to DRG cultures, and responses from se-lected neurons are shown in Fig. 2 A–C.We used the protocol shown in Fig. 2 A–C and compiled data

for the cellular responses of 2,026 mouse lumbar DRG neurons(Table 1). The fraction of cells that responded varied consider-ably from one challenge compound to the next challenge com-pound. A large proportion of the cells responded to capsaicin(39%), mustard oil [allyl isothiocyanate (AITC; 32%)], or ATP(76%), but only a minor fraction (<10%) responded to menthol,histamine, or acetylcholine (ACh) under these experimentalconditions. We refer to experiments using the class of com-pounds that activate only a minor fraction of the DRG cells asdiagnostic RA challenges.

Definition of DRG Neuron Subclasses Using Diagnostic RA Challenges:Histamine, ACh, and Menthol.We used diagnostic RA challenges asa first step to identify a neuronal subtype. DRG neuron sub-classes that responded to diagnostic RA challenge compoundscould be further subdivided by their responses to other challengecompounds. We provide a few examples below. One example isthe small percentage of cells that responded to histamine. Ap-proximately one-half of the histamine-responsive neurons hadthe same phenotypic profile: these cells did not respond to theother diagnostic RA challenge compounds (ACh and menthol)or AITC but did respond to capsaicin and ATP (Fig. 2B andTable 2). The average area of the cell soma (cell area) for thisclass of neurons, 240 μm2, was relatively small.One putative subtype of ACh-responsive cells did not respond

to any of the other five challenge compounds (Fig. 2A and Table2). Furthermore, none of those cells stained with Alexa-fluor-568–labeled IB4 (Fig. S1 and Table 3). A different subclass ofACh-responsive cells responded to capsaicin and ATP but not tomenthol, histamine, or AITC (Fig. S1 and Table 2), and themajority of these cells stained with IB4 (Fig. S1 and Table 3).The ACh-sensitive cells that did not respond to any other chal-lenge compound were predominantly large cells (average cellarea = 670 μm2); in contrast, the subset of ACh-responsive cells

Fig. 1. Images of dissociated mouse lumbar DRG neurons loaded with Fura-2-AM dye. A–D are images of the same field of view. Fluorescence imageswere acquired as described inMaterials and Methods. (A) Bright-field image.(B) Fluorescence image acquired with 380-nm excitation and 510-nm emis-sion filters. (C) Pseudocolored ratiometric calcium image obtained undercontrol conditions (i.e., before depolarization). The color scale indicates thatthe resting cytoplasmic calcium concentration is relatively low (magenta andblue). (D) Pseudocolored ratiometric image obtained on depolarization ofthe neurons by 100 mM KCl. The color scale, same as the color scale in C,indicates that the cytoplasmic calcium concentration is relatively high inmany cells (green, yellow, and red). (Scale bar: 30 μm.) Glial cells did notrespond to 100 mM KCl with elevated cytoplasmic calcium, presumably be-cause they lack voltage-gated calcium channels.

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that were also capsaicin- and ATP-sensitive was substantiallysmaller (average cell area = 270 μm2) (Fig. 3 and Table 2).Menthol-sensitive DRG neurons could also be subdivided on

the basis of their responsiveness to other compounds. For ex-ample, a subset of menthol-sensitive DRG neurons was alsosensitive to capsaicin, which other investigators have observed(13, 15, 21–23). A different putative DRG subtype respondedexclusively to menthol (Fig. 2C and Table 2) and did not stainwith IB4 (Table 3). On average, they were exceptionally smallcells (average cell area = 150 μm2) (Table 2) that displayedunusually noisy and variable resting [Ca2+]i (Fig. 2C). Part of thevariability was characterized by a transient dip in the [Ca2+]ibaseline each time the static bath solution was replaced witha bath solution containing an RA challenge compound, with theexception of menthol or KCl, which elicited increases in [Ca2+]i(Fig. 2C). The size distribution of this class of menthol-sensitivecells is shown in Fig. 3. Another putative DRG subtyperesponded to menthol, AITC, and ATP (Fig. 2C and Table 2)and stained with IB4 (Table 3). On average, they were largercells than those cells that responded only to menthol (averagecell area = 270 μm2) (Table 2).

MP Challenge Protocol. Each neuron has molecular componentswith activation that promotes depolarization (e.g., voltage-gatedNa and Ca channels) or hyperpolarization (e.g., voltage-gated Kchannels). On application of a depolarizing stimulus, the in-crease in [Ca2+]i is determined by a balance between the actionsof these two sets of components. Thus, the application of an MPchallenge compound can be used to assess whether particularvoltage-sensitive ion channels are functionally expressed in theplasma membrane of a neuron by monitoring a decrease or in-crease in the magnitude and/or kinetics of the calcium signalelicited on membrane depolarization (e.g., by elevating extra-cellular K+, [K+]o).Calcium signals were elicited by depolarizing neurons with 25-

mM KCl pulses before and after application of an MP challengecompound, which is described in Materials and Methods andshown in Fig. 4. To assess how different DRG cells respond tovarious MP challenge compounds, we first applied two classicalpharmacological agents: tetrodotoxin (TTX; it inhibits a broadspectrum of voltage-gated Na channels) and tetraethylammo-nium (TEA; a standard wide-spectrum, voltage-gated K-channelblocker). As expected, they produced opposite effects: TTXdecreased the calcium signal in response to a standard KCl pulse,whereas TEA increased it, as described below.

Block by TTX. The reduction by TTX of the high [K+]o-inducedcalcium signal varied from cell to cell, which is shown in Fig. 5 A–D. We expected the application of KCl (25 mM) to activatevoltage-gated Na channels by moderately depolarizing the cells.Our hypothesis was that the opening of voltage-gated Na chan-nels would be required to further depolarize the membranesufficiently to activate high-voltage–activated Ca channels.However, TTX significantly reduced (by >3 SDs below the meaninternal control value) the height of each peak in only a smallsubset of cells (<20%), with the high [K+]o-induced calciumsignals of most cells largely nonresponsive to TTX (Fig. 5E).

Diverse Responses to TEA. TEA, at 25 mM, has been shown toblock most of the TEA-sensitive (sustained) K currents in small-diameter rat DRG neurons without blocking A-type currents(24). In a separate study, 5 mM TEA applied to current-clam-ped, small-diameter rat DRG neurons depolarized the mem-brane, reduced the threshold for action potential generation, andextended the duration of an action potential (25). In view ofthese studies, we used TEA at both 25 and 5 mM concentrations.A surprising variety of responses were induced by TEA. The

same types of effects were elicited with either 25 or 5 mM TEA.

Fig. 2. Example calcium-imaging traces from RA challenges. Each trace is asingle neuron’s response to the challenge compounds indicated at the bottomof each panel. The x axis is the same for all traces in a given panel. The y axis(same for Figs. 4–6) is a relative measure of [Ca2+]i determined by the 340/380nm excitation ratio described inMaterials andMethods. Challenge compoundswere ACh, 1 mM acetylcholine; ATP, 10 μM adenosine 5′-triphosphate; AITC,200 μMallyl isothiocyanate; C, 300nMcapsaicin; H, 50 μMhistamine;M, 200 μMmenthol; K, 100 mM KCl (A–C) or 25 mM KCl (D); and TEA, 10 mM tetraethy-lammonium chloride. Arrows indicate when each compound was applied tothe bath. (A–C) Typical sequence of RA challenges. Challenge compounds wereapplied at 5-min intervals for ∼15 s. ACh was applied two times to show re-producibility of responses. KCl (100 mM) was applied at the end of the series,and therefore, nonresponsive cells could be excluded from additional analysis.The maximum response to KCl was cropped for some traces, and therefore,heights of lesser peaks would be more evident. (C and D) ATP, menthol, andAITC were used to differentiate between neurons only sensitive to mentholand neurons sensitive to all three challenge compounds, which are highlightedin blue. (D) Responses from one neuron of each type are shown. Neurons werealso depolarized by KCl (25 mM) pulses at 7-min intervals before and afterapplication of 10 mM TEA (indicated by horizontal bar), a blocker of voltage-gated K channels, to compare TEA with KCl-elicited responses. The red color isfor emphasis only. It highlights the different types of responses to TEA.

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Some DRG neurons responded directly to TEA addition withsustained, elevated [Ca2+]i and/or repetitive [Ca2+]i spikes ofvariable intermittency (Fig. 5 F and G, respectively). In additionto these direct effects, many DRG neurons responded to TEAonly with an enhanced response to the subsequent KCl pulse(Fig. 5 H and I). The enhancement took the form of increasedpeak height, width, or both. In all cells, the effects of TEA werereadily reversible. A subset of cells was unresponsive to TEA;their high [K+]o-induced calcium signals were unaffected byTEA (Fig. 5J).

Definition of DRG Neuron Subclasses Using Diagnostic MP Challenges:κM-Conopeptide RIIIJ, Dendrotoxin-K, and Conopeptide pl14a. Asnoted above for TEA, K-channel blockers may elicit differenttypes of phenotypic responses in different cells. Some neuronsresponded directly to the challenge compound. This responsetypically occurred only in a small proportion of neurons andcould be used to define subclasses in the same way as the di-agnostic RA challenge compounds described above.The addition of 1 μM κM-conopeptide RIIIJ (κM-RIIIJ),

a conopeptide previously reported to be highly selective forKV1.2-containing channels (26), caused a direct response withsustained, elevated [Ca2+]i in a fraction of large-diameter cells(Fig. 6). Large-diameter neurons with definitive direct responsesto κM-RIIIJ did not respond to ACh (Fig. 6A). Thus, they de-lineate a different subclass of cells from the ACh-responsive,large-diameter cells described above.In addition to κM-RIIIJ, two other selective K-channel

antagonists are reported to target voltage-gated K channels inthe same subfamily (shaker or KV1). One of these antagonists,a conopeptide from Conus planorbis (pl14a), is selective forKV1.6 (27), whereas a kunitz domain small protein from mambavenom, Dendrotoxin-K (Dtx-K), is reported to be highly selec-tive for KV1.1 (28). In previous reports, the targeting selectivitiesof κM-RIIIJ, pl14a, and Dtx-K were assessed by tests on heter-ologously expressed homomeric channels (26–28).

Here, we compared the direct responses to pl14a and Dtx-Kwith the direct responses to κM-RIIIJ to evaluate the overlapbetween the neurons responsive to pl14a and Dtx-K with thelarge-diameter neurons that directly respond to κM-RIIIJ. Forthe large-diameter DRG neurons, the response to κM-RIIIJ wasinversely correlated with the response to pl14a (Fig. 6B). Forneurons in which κM-RIIIJ produced robust direct effects, pl14aproduced no direct effects and vice versa. Even more striking wasthe fact that those cells that did respond directly to either agenthad different phenotypes. Thus, as illustrated in Fig. 6B, thesmooth, sustained rise in [Ca2+]i observed with κM-RIIIJ wasstrikingly different from the spiky response to pl14a. In contrast,there was considerable overlap between the cells that respondeddirectly to Dtx-K and the cells that responded directly to κM-RIIIJ (Fig. 6C). The direct response of most cells to Dtx-K wassimilar in phenotype to the response observed for κM-RIIIJ (i.e.,a smooth, sustained increase in [Ca2+]i).Similar to the variability in responses to TEA, some DRG

neurons responded to κM-RIIIJ only indirectly on elevation of[K+]o (Fig. S2B). Such cells were larger in diameter, on average(∼480 μm2), than the histamine- or menthol-sensitive cells (av-erage cell sizes ≤ 270 μm2) (Table 2). Thus, the indirect responsesconstitute another functional phenotype that also subdividesDRG neurons.A noteworthy feature of experiments with Dtx-K was the dif-

ferential reversibility observed among the responsive neurons(Fig. S3). In some neurons, the effect of Dtx-K was rapidly re-versed on washout (Fig. S3C), whereas in other neurons, thetoxin’s effect was either slowly reversible or almost irreversible(Fig. S3 D and E).Although a moderate fraction of the total number of cells

responded to selective K-channel inhibitors, those cells that re-spond with characteristic functional phenotypes, which are il-lustrated in Figs. 5 and 6 and Figs. S2 and S3, comprise only asmall, coherent subset of neurons with other correlated pheno-typic properties. Accordingly, the experiments described aboveshow that a specific type of phenotypic response to selectiveK-channel antagonists can be used as a diagnostic MP challenge.

Additional Characterization of DRG Neuron Subtypes by CombiningRA and MP Challenges. The approach that we used to defineneuronal subclasses also provides opportunities for their furtherfunctional characterization. The menthol-sensitive cells de-scribed above can be easily identified in the population of DRGneuronal subtypes. In the experiment shown in Fig. 2D, ATP,menthol, and AITC were applied to differentiate between twoputative subtypes of menthol-sensitive neurons. The menthol-sensitive subtype that was insensitive to other RA challenges wasresponsive to TEA, an MP challenge; furthermore, in each in-stance, a characteristic spiky response to TEA was observed. Incontrast, a spiky response to TEA was never observed in the cellsthat were sensitive to menthol, ATP, and AITC. Instead, thosecells responded either only indirectly upon high [K+]o-induced

Table 1. Percentage of mouse lumbar DRG neurons thatresponded to RA challenge compounds

Challenge compound Number of responsive cells Cells (%)

ACh (1 mM) 158 7.8ATP (10 μM) 1,536 75.8Histamine (50 μM) 131 6.4Menthol (200 μM) 178 8.8AITC (mustard oil; 200 μM) 651 32.1Capsaicin (300 nM) 791 39.0KCl (100 mM) 2,026 100.0

Only data from viable neurons, defined by their ability to respond to 100mM KCl at the end of the trial, are presented in this table and all subse-quent tables.

Table 2. Selected subclasses of mouse lumbar DRG neurons defined by RA challenges

Diagnostic RA challenge compound Profiling compounds Distinctivefunctionalphenotype*

Averagecell size(μm2)

Numberof cells†ACh Menthol Histamine Cap AITC ATP

+ − − − − − 670 28+ − − + − + 270 30− + − − − − + 150 33− + − − + + 270 24− − + + − + 240 65

*This phenotype is characterized by highly variable resting [Ca2+]i.†Each neuron subclass represents a very small proportion (1.2–3.2%) of the total of 2,026 neurons examined.

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depolarization or directly to TEA with a smooth, sustained in-crease in [Ca2+]i (Fig. 2D).Dtx-K, κM-RIIIJ, and pl14a were also tested after application

of menthol. The menthol-sensitive neurons that did not respondto other RA challenges were also resistant to all of these MPchallenges, suggesting that inhibition of a different subset ofvoltage-gated K channels (non-KV1 family sensitive to TEA) isresponsible for initiating the spiky response to TEA observed inFig. 2D. Thus, after a neuronal subtype has been identified bya set of markers, it may be further characterized by additionalRA and MP challenge compounds.In Table 3, we summarize the data obtained for seven selected

subclasses of DRG neurons that were defined by diagnostic RAand/or MP challenges, cell size, and IB4 staining. Some of thesesubclasses probably conform to our narrow definition of DRGsubtype because of their highly consistent cross-correlation withmultiple functional and structural markers.

DiscussionFunctional Profiling. This report describes experiments performedon mouse DRG neurons in which we used a variety of functionalmarkers to define several distinctive neuronal subclasses. Thisanalysis has parallels to the early anatomical characterization ofneurons; the classes first recognized by Ramón y Cajal werethose classes with the most striking morphology. Using our ap-proach, DRG neuronal subclasses that exhibit the most distinc-tive functional properties are the easiest to define. Thus, we havechosen to highlight a few neuronal subclasses with clearly dis-tinctive functional properties. A future objective is to use ourexperimental approach, coupled with cluster analysis, to developa taxonomy of all neuronal subtypes within the DRG.

Based on cross-correlations of multiple markers, we haveidentified a few subclasses of DRG neurons, which are summa-rized in Table 3. These subclasses include one histamine-sensi-tive, two ACh-sensitive, and two menthol-sensitive as well aslarge-diameter neurons that responded directly to κM-RIIIJ andmedium-diameter neurons that responded indirectly to κM-RIIIJ. These neurons comprise only a small minority (∼15%altogether) of all of the different cells in the culture. Some of thesubclasses also showed highly consistent cross-correlations withseveral markers (e.g., consistent responses to functional markersand consistent IB4 staining). Thus, they probably conform to ournarrow definition of neuronal subtypes, with specific commonphysiological functions. However, many more DRG subtypesremain to be identified, and all of the subtypes require addi-tional characterization.

Menthol-Sensitive Neurons. Two putative subtypes of menthol-sensitive neurons that we have described in this paper are dis-tinguished from each other by strikingly divergent functionalphenotypes. Only one subtype responded to AITC and ATP andstained with IB4, which we will call the MA+ subtype (i.e.positive responses to menthol, ATP and AITC). The othersubtype was exceptionally small, IB4-negative, and did not re-spond to any other RA challenge compounds; these cells alsodisplayed highly variable resting [Ca2+]i (Fig. 2C). We will callthis subtype the M+ subtype (i.e., exclusively menthol-positive).The facile identification of the M+ and MA+ subtypes should

allow us to begin to systematically characterize their cellularneurophamacology. For example, we can investigate the sourceof variability in [Ca2+]i observed in the M+ subtype, which maybe caused by activity of TRPM8 at room temperature. Traces inFig. 2C show that there was a transient dip in the [Ca2+]i

Table 3. Seven selected subclasses of mouse lumbar DRG neurons defined by RA and/or MP challenges

DRG neuronsubclass Examples

Averagecell size (μm2)

Positive functional markers(implicated protein expression)

Negative functionalmarkers IB4 stain

CulturedDRG neurons

(%)

Large1 Fig. 6 680 Direct κM-RIIIJ+* (KV1.2) ACh−, pl14a− IB4− 2.3

Direct Dtx-K+ (KV1.1)2 Figs. 2A, 3A, and 6A 670 ACh+ (AChRs) Men−, Hist−, Cap−,

AITC−, ATP−IB4− 1.4

Medium3 Fig. S2 480 Indirect κM-RIIIJ+† (KV1.2) IB4+/− 4.1‡

Small4 Fig. 3B and S1 270 ACh+ (AChRs) Men−, Hist−, AITC− IB4+/− 1.5

Cap+ (TRPV1)ATP+ (P2X/Y)

5 Fig. 2B 240 Hist+ (HistRs) ACh−, Men−, AITC− IB4+/− 3.2Cap+ (TRPV1)ATP+ (P2X/Y)

6 Fig. 2 C and D 270 Men+ (TRPM8) ACh−, Hist−, Cap− IB4+ 1.2AITC+ (TRPA1)ATP+ (P2X/Y)TEA sustained response

7 Figs. 2 C and D and 3C 150 Men+ (TRPM8) ACh−, Hist−, Cap−,AITC−, ATP−

IB4− 1.6TEA spiky response

ACh, 1 mM acetylcholine; AChRs, acetylcholine receptors; AITC, 200 μM allyl isothiocyanate; ATP, 10 μM adenosine 5′-triphosphate; Cap, 300 nM capsaicin;Dtx-K, 100 nM Dendrotoxin-K; Hist, 50 μM histamine; HistRs, histamine receptors; IB4, isolectin B4; IB4+/−, mix of IB4+ and IB4−; κM-RIIIJ, 1 μM κM-conopeptideRIIIJ; KV1, KV1 potassium channels; Men, 200 μM menthol; P2X/Y, P2X or P2Y receptors; pl14a, 16 μM conopeptide pl14a; TEA, 10 mM tetraethylammoniumchloride; TRP (A1, M8, and V1), transient receptor potential channels; +, responsiveness to the compound; −, lack of responsiveness to the compound.*κM-RIIIJ directly elicited a sustained calcium signal.†κM-RIIIJ indirectly amplified the high [K+]o-elicited response. Indirect effects by κM-RIIIJ were scored if there was an increase in the [K+]o-elicited peak height(calcium signal) >3 SDs over the peak height of the average [K+]o-elicited peak height (control calcium signal) before application of κM-RIIIJ.‡Only medium-sized neurons with cell areas between 300 and 600 μm2 were included in this percentage. However, the calculation for the average cell area of480 μm2 for subclass 3 included neurons of all diameters that responded indirectly to κM-RIIIJ.

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baseline of the M+ cells each time the static bath solution wasreplaced (except menthol- and KCl-elicited increases in [Ca2+]i).Each dip may be caused by a slight warming on addition of so-lution to a slightly cool background produced by evaporativecooling of the static bath. Presumably, the warming reducesTRPM8 activity (i.e., fewer channels open), causing the dip,followed by evaporative cooling, which increases TRPM8 activityand thus, restores the [Ca2+]i baseline after each dip. Some ofthe baseline variability may also be related to action potentialbursting observed for some cold-sensitive units (29) and/or ac-tivity of ryanodine receptors, etc.By using additional challenge compounds and experimental

protocols, it should be feasible to identify the molecular isoformsof various ion channels and receptors present in each menthol-sensitive subtype. For instance, what voltage-gated Ca-channelisoforms are present, and do they differ between the M+ andMA+ subtypes? We can even begin to address which K channelsare present in each subtype and determine whether homomericor heteromeric channels are responsible for the functional phe-notypes observed. The constellation of such receptors and ionchannels in different neuronal subtypes constitute the molecularbasis of their divergent functional properties. Thus, the cellularneuropharmacology of the menthol-sensitive subtypes shouldprovide a far more refined characterization of these cells.The menthol receptor, TRPM8, is strongly implicated in cold

sensing (21, 30), but the precise physiological roles of the dif-ferent menthol-sensitive DRG subtypes are less clear. As eachneuronal subtype is further characterized, the properties un-covered may provide a guide to their physiological roles in vivo.For example, it is apparent from the divergent phenotypes eli-cited by TEA (Fig. 2D) that the complement of voltage-gated K

channels in the two subtypes of menthol-sensitive cells likelydiffer. Notably, other investigators have shown that sensory tri-geminal neurons responsive to innocuous cool temperatures(low-threshold cold thermoreceptors) show high expression ofTRPM8 and low expression of KV1 channels, whereas neuronsresponsive to noxious cold temperatures (high-threshold coldthermoreceptors) show low expression of TRPM8 and high ex-pression of KV1 channels, with KV1 acting as an excitabilitybrake in high-threshold, cold-sensitive neurons (14).In our experiments, the M+ subtype showed robust menthol

responses (Fig. 2C) (suggesting high TRPM8 expression), in-sensitivity to KV1 blockers (suggesting low KV1 expression), andinstability in [Ca2+]i at room temperature, all consistent with thehypothesis that the M+ subtype is a low-threshold cold ther-moreceptor. In contrast, the MA+ subtype typically respondedrelatively weakly to menthol (Fig. 2C) (suggesting relatively lowTRPM8 expression), whereas the responses to ATP (31–33) andAITC are implicated in nociception; the AITC receptor,TRPA1, is implicated in noxious cold nociception (34–38). Thesedata, coupled with the relatively stable [Ca2+]i at room tem-perature, are all consistent with the hypothesis that the MA+subtype is a high-threshold cold thermoreceptor. We have ob-tained preliminary temperature-sensitivity data for the M+ andMA+ neurons in support of these hypotheses, and a detailedcharacterization of these two DRG subtypes is presently beingcarried out.

K-Channel Antagonists as Challenge Compounds. In the experimentsdescribed above, a variety of K-channel antagonists wereused. The most extensively analyzed was κM-RIIIJ, reportedto have high selectivity for KV1.2 compared with other homo-meric voltage-gated K channels. A distinctive subclass of large-diameter cells responded directly to this conopeptide, dis-tinguishing this subset of large-diameter neurons from thesubclass sensitive only to ACh (Fig. 6A).In addition to κM-RIIIJ, two other relatively selective K-

channel antagonists were used, the pl14a conopeptide and Dtx-

Fig. 3. Size distributions of neurons responsive to select RA challengecompounds. Data are shown for only a few neuronal subclasses. The x axis isthe same for all panels. Cell area is the area of a cross-section of the cellsoma. (A) ACh only refers to neurons that responded to ACh but not to anyother RA challenge compounds. (B) ACh + ATP + Cap refers to neurons thatresponded to ACh, ATP, and capsaicin but no other RA challenge com-pounds. (C) Menthol only refers to neurons that responded to menthol butno other RA challenge compound.

Fig. 4. MP challenge protocol illustrated with calcium-imaging traces. Eachtrace represents a single neuron’s response. Each peak in a trace is the re-sponse to a 25-mM KCl pulse applied at 7-min intervals. The duration of eachKCl pulse (∼15 s) is indicated by the vertical bars (1–8). The first four KClpulses (1–4) served as internal controls to monitor the variability in the eli-cited calcium signals. The last three KCl pulses (6–8) allowed us to monitorthe reversibility of a compound’s effects. The compound of interest wasapplied at minute 23 and remained in the bath for 6 min, which is indicatedby the horizontal bar in Upper. The fifth KCl pulse, at minute 29 (peak 5),shows that the compound caused an amplification of the high [K+]o-elicitedcalcium signal, which was readily reversible (indicated by peaks 6–8). (Lower)Vehicle trials, in which only observation solution was applied to the bath atminute 23, served as external controls. The red color is for emphasis only. Ithighlights the difference between a response to a compound (Upper; com-pound trial) and a control without a compound (Lower; vehicle trial). At theend of the experiment, at minute 57, 300 nM capsaicin was applied for 1 min(black circles).

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K, and they were reported to have high selectivity for KV1.6 andKV1.1, respectively. It is clear that the subset of neurons thatresponded directly to pl14a comprises a different neuronal sub-class from the large neurons that responded directly to κM-RIIIJ. Although fewer cells were analyzed, the neurons thatresponded directly to pl14a were, on average, smaller in size.Thus, different subsets of neurons presumably have inverselyproportional expression levels of KV1.2 and KV1.6. In addition,the phenotypes elicited by the two conopeptides were different:a subset of DRG neurons responded to κM-RIIIJ with a smooth,sustained increase in [Ca2+]i, whereas a spiky response was ob-served in the neurons that responded directly to pl14a (Fig. 6B).In contrast, there was considerable overlap in DRG neurons thatresponded to κM-RIIIJ and Dtx-K when direct responders werescored (Fig. 6C). The results suggest that the KV1.1 subunitoverlaps significantly with KV1.2 in large-diameter DRG neu-rons, consistent with previous expression studies (24).The results using Dtx-K are particularly notable; in some

neurons, the effects of Dtx-K were rapidly reversible but onlyslowly reversible in other neurons (Fig. S3), suggesting that Dtx-K may block different heteromers of KV1.1/1.X with variableaffinity in different neuronal subtypes.

Prospectives. The experiments using ACh or K-channel antago-nists as challenge compounds provide some insight into an im-portant long-term direction of the cellular neuropharmacologicalapproach described in this work. K channels and nicotinic ace-tylcholine receptors (nAChRs) are examples of ion channels thatendow the nervous system with functional complexity at the levelof individual macromolecules, because the functional receptor/ion channel is multimeric. Although there is a limited number ofsubunit genes, the subunits can combine into heteromers, mak-ing an enormous array of different combinations possible. Forsuch ion channel families, it has been challenging to identify thefunctional roles for the individual molecular isoforms. Thestandard approach to identify function is gene KOs. The prob-lem for multimeric complexes, such as the K-channel or nAChRfamilies, is that KO of a single subunit does not just abolish oneisoform but all potential heteromeric combinations containingthe subunit encoded by the KO gene. This problem leads toa complex phenotype that does not reveal the function of anyindividual molecular isoform but rather, is the result of ablatingall isoforms that contain that subunit.The platform that we have developed makes it possible, in

principle, to identify particular neuronal subtypes that function-

ally express specific nAChR- and K-channel isoforms. With theappropriate neuropharmacological tools (e.g., the α- or κ-con-opeptides among other reagents), the molecular isoform(s)

Fig. 5. Selected calcium-imaging traces from MPchallenges. Each trace represents a single neu-ron’s response. These data were obtained by us-ing the MP challenge protocol exemplified by Fig.4; x axis is the same for all traces, and the redcolor is for emphasis only. It highlights the dif-ferent types of responses to each challengecompound. (A–E) Effects of 1 μM TTX, which wasapplied for 6 min starting at minute 23 (indicatedby horizontal bars). (F–J) Effects of 25 mM TEA,which was applied for 6 min starting at minute 23(bars). At the end of the experiment, 300 nMcapsaicin was applied for 1 min at minute 57(black circles).

Fig. 6. Selected traces from large-diameter neurons (cell area > 600 μm2)illustrating the effects of voltage-gated K-channel blockers. Each trace rep-resents a single neuron’s response. The x axis is the same for all traces ina given panel. Challenge compounds were ACh, 1 mM acetylcholine; C, 300nM capsaicin; K, 25 mM KCl; pl14a, 16 μM conopeptide pl14a; RIIIJ, 1 μM κM-conopeptide RIIIJ; and Dtx, 100 nM Dendrotoxin-K. Arrows and bars indicatewhen each challenge compound or KCl was applied; the latter was applied at7-min intervals except as noted. The red color is for emphasis only. It high-lights differences in responsiveness to the challenge compounds used. (A)ACh was applied (at minute 1) to identify responders (blue is for emphasisonly), after which time the KCl pulses were given before and after applica-tion of κM-RIIIJ (bar). These neurons were resistant to capsaicin, which istypical for large-diameter DRG neurons. (B) KCl pulses presented before andafter application of κM-RIIIJ (first bar) and conopeptide pl14a (second bar).These neurons were resistant to capsaicin. (C) KCl pulses applied before andafter application of κM-RIIIJ (first bar) and Dtx-K (second bar).

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expressed in specific neuronal subtypes can be identified. There-fore, this identification opens the door to characterize the func-tional role of a specific receptor/ion channel isoform in the relevantneuronal circuitry. If one knows the molecular isoform(s)expressed in a specific neuron, inhibition of each molecular iso-form should allow an assessment of the change in the properties ofthe circuit pertaining to that cell. In this way, the cellular neuro-pharmacology of individual neuronal subtypes provides a potentialbridging technology between systems and molecular neuroscience.

Materials and MethodsMouse DRG Dissection and Cell Culture. Detailed procedures are provided inSI Materials and Methods. Briefly, WT C57BL/6 mice between the ages of20–30 d postnatal were euthanized with CO2 just before the dissection ofDRGs. Dissection, cell dissociation, and cell culture methods were essentiallyas reported previously (39), except the minimal essential media contained10 mM Hepes and glial-derived neurotrophic factor was used at a final con-centration of 20 ng/mL.

Calcium Imaging. Imaging was performed as detailed in SI Materials andMethods. Briefly, cells loaded with Fura-2 dye were excited intermittentlywith 340- and 380-nm light, whereas fluorescence emission was monitoredat 510 nm. An image was captured at each excitation wavelength and the340/380 nm ratio of fluorescence intensity was acquired (usually) one timeper second to monitor the relative changes in [Ca2+]i for each cell over time.Thus, we obtained the 340/380-nm ratiometric images shown in Fig. 1 andthe 340/380-nm ratiometric data (traces) shown in Figs. 2–6. In a given

experiment, >100 cells were individually imaged simultaneously. Increases in[Ca2+]i were elicited by ∼15-s application of various RA challenge compoundsor elevated [K+]o in observation solution. MP challenge compounds in ob-servation solution were applied to cells for time periods indicated in Figs. 1–6. For RA challenge compounds, we generally chose high concentrations toavoid issues of dose response. The concentration of ACh was chosen to besaturating or nearly saturating for all subtypes of ACh receptors. We appliedATP at the high end of its expected physiological concentration (in muscleinterstitium) (39). The other RA challenge compound concentrations areconsistent with common literature values that have been used to identifyresponsive sensory neurons previously (13–15, 19, 35). For MP challengecompounds, we chose either high concentrations to obtain broad-spectrumblock of K or Na channels (i.e., TEA and TTX, respectively) or relatively lowconcentrations that were expected to provide subtype-selective block ofparticular K-channel subtypes [i.e., Dtx-K (KV1.1) (28), κM-RIIIJ (KV1.2) (26),and conopeptide Pl14a (KV1.6) (27)]. After calcium imaging, cells werestained with Alex-fluor-568–labeled IB4 and imaged. Data for each experi-ment were screened manually for cells that did not respond to elevated [K+],washed out of the field of view, or produced irreversible high [Ca2+]i duringthe experiment. Such cells were excluded from additional analysis.

ACKNOWLEDGMENTS. We thank Ron Hughen and Robert J. Malcolm fortheir advice in the early stages of this work and Lita Imperial for synthesizingconopeptide pl14a. S.R. acknowledges the Desh Videsh Fund of ShanmughaArts, Science, Technology & Research Academy (SASTRA) University. Thiswork was supported by National Institute of General Medical SciencesGrant GM48677.

1. Comer CM, Robertson RM (2001) Identified nerve cells and insect behavior. ProgNeurobiol 63:409–439.

2. Lu SG, Zhang X, Gold MS (2006) Intracellular calcium regulation among sub-populations of rat dorsal root ganglion neurons. J Physiol 577:169–190.

3. Hobert O, Carrera I, Stefanakis N (2010) The molecular and gene regulatory signatureof a neuron. Trends Neurosci 33:435–445.

4. Nelson SB, Hempel C, Sugino K (2006) Probing the transcriptome of neuronal celltypes. Curr Opin Neurobiol 16:571–576.

5. Nelson SB, Sugino K, Hempel CM (2006) The problem of neuronal cell types: Aphysiological genomics approach. Trends Neurosci 29:339–345.

6. López-Muñoz F, Boya J, Alamo C (2006) Neuron theory, the cornerstone of neuro-science, on the centenary of the Nobel Prize award to Santiago Ramón y Cajal. BrainRes Bull 70:391–405.

7. Franceschetti S, Sancini G, Panzica F, Radici C, Avanzini G (1998) Postnatal differen-tiation of firing properties and morphological characteristics in layer V pyramidalneurons of the sensorimotor cortex. Neuroscience 83:1013–1024.

8. Bernard A, Sorensen SA, Lein ES (2009) Shifting the paradigm: New approaches forcharacterizing and classifying neurons. Curr Opin Neurobiol 19:530–536.

9. Sugino K, et al. (2006) Molecular taxonomy of major neuronal classes in the adultmouse forebrain. Nat Neurosci 9:99–107.

10. Shoemaker LD, Arlotta P (2010) Untangling the cortex: Advances in understandingspecification and differentiation of corticospinal motor neurons. Bioessays 32:197–206.

11. Molyneaux BJ, Arlotta P, Menezes JR, Macklis JD (2007) Neuronal subtype specifica-tion in the cerebral cortex. Nat Rev Neurosci 8:427–437.

12. Caterina MJ, Julius D (2001) The vanilloid receptor: A molecular gateway to the painpathway. Annu Rev Neurosci 24:487–517.

13. Hjerling-Leffler J, Alqatari M, Ernfors P, Koltzenburg M (2007) Emergence of func-tional sensory subtypes as defined by transient receptor potential channel expression.J Neurosci 27:2435–2443.

14. Madrid R, de la Peña E, Donovan-Rodriguez T, Belmonte C, Viana F (2009) Variablethreshold of trigeminal cold-thermosensitive neurons is determined by a balancebetween TRPM8 and Kv1 potassium channels. J Neurosci 29:3120–3131.

15. Xing H, Ling J, Chen M, Gu JG (2006) Chemical and cold sensitivity of two distinctpopulations of TRPM8-expressing somatosensory neurons. J Neurophysiol 95:1221–1230.

16. Persson AK, Xu XJ, Wiesenfeld-Hallin Z, Devor M, Fried K (2010) Expression of DRGcandidate pain molecules after nerve injury—a comparative study among five inbredmouse strains with contrasting pain phenotypes. J Peripher Nerv Syst 15:26–39.

17. Caterina MJ, et al. (1997) The capsaicin receptor: A heat-activated ion channel in thepain pathway. Nature 389:816–824.

18. Alving K, et al. (1991) Association between histamine-containing mast cells andsensory nerves in the skin and airways of control and capsaicin-treated pigs. CellTissue Res 264:529–538.

19. Liu Q, et al. (2009) Sensory neuron-specific GPCR Mrgprs are itch receptors mediatingchloroquine-induced pruritus. Cell 139:1353–1365.

20. Shim WS, Oh U (2008) Histamine-induced itch and its relationship with pain. Mol Pain4:29.

21. McKemy DD, Neuhausser WM, Julius D (2002) Identification of a cold receptor revealsa general role for TRP channels in thermosensation. Nature 416:52–58.

22. Reid G, Babes A, Pluteanu F (2002) A cold- and menthol-activated current in rat dorsalroot ganglion neurones: Properties and role in cold transduction. J Physiol 545:595–614.

23. Viana F, de la Peña E, Belmonte C (2002) Specificity of cold thermotransduction isdetermined by differential ionic channel expression. Nat Neurosci 5:254–260.

24. Vydyanathan A, Wu ZZ, Chen SR, Pan HL (2005) A-type voltage-gated K+ currentsinfluence firing properties of isolectin B4-positive but not isolectin B4-negative pri-mary sensory neurons. J Neurophysiol 93:3401–3409.

25. Safronov BV, Bischoff U, Vogel W (1996) Single voltage-gated K+ channels and theirfunctions in small dorsal root ganglion neurones of rat. J Physiol 493:393–408.

26. Chen P, Dendorfer A, Finol-Urdaneta RK, Terlau H, Olivera BM (2010) Biochemicalcharacterization of kappaM-RIIIJ, a Kv1.2 channel blocker: Evaluation of car-dioprotective effects of kappaM-conotoxins. J Biol Chem 285:14882–14889.

27. Imperial JS, et al. (2006) A novel conotoxin inhibitor of Kv1.6 channel and nAChRsubtypes defines a new superfamily of conotoxins. Biochemistry 45:8331–8340.

28. Robertson B, Owen D, Stow J, Butler C, Newland C (1996) Novel effects of den-drotoxin homologues on subtypes of mammalian Kv1 potassium channels expressedin Xenopus oocytes. FEBS Lett 383:26–30.

29. Braun HA, Bade H, Hensel H (1980) Static and dynamic discharge patterns of burstingcold fibers related to hypothetical receptor mechanisms. Pflugers Arch 386:1–9.

30. Peier AM, et al. (2002) A TRP channel that senses cold stimuli and menthol. Cell 108:705–715.

31. Cockayne DA, et al. (2005) P2X2 knockout mice and P2X2/P2X3 double knockout micereveal a role for the P2X2 receptor subunit in mediating multiple sensory effects ofATP. J Physiol 567:621–639.

32. Cockayne DA, et al. (2000) Urinary bladder hyporeflexia and reduced pain-relatedbehaviour in P2X3-deficient mice. Nature 407:1011–1015.

33. Ou S, et al. (2011) Effect of lappaconitine on neuropathic pain mediated by P2X3receptor in rat dorsal root ganglion. Neurochem Int 58:564–573.

34. Bandell M, et al. (2004) Noxious cold ion channel TRPA1 is activated by pungentcompounds and bradykinin. Neuron 41:849–857.

35. Bautista DM, et al. (2006) TRPA1 mediates the inflammatory actions of environmentalirritants and proalgesic agents. Cell 124:1269–1282.

36. Jordt SE, et al. (2004) Mustard oils and cannabinoids excite sensory nerve fibresthrough the TRP channel ANKTM1. Nature 427:260–265.

37. Kwan KY, et al. (2006) TRPA1 contributes to cold, mechanical, and chemical noci-ception but is not essential for hair-cell transduction. Neuron 50:277–289.

38. Story GM, et al. (2003) ANKTM1, a TRP-like channel expressed in nociceptive neurons,is activated by cold temperatures. Cell 112:819–829.

39. Light AR, et al. (2008) Dorsal root ganglion neurons innervating skeletal muscle re-spond to physiological combinations of protons, ATP, and lactate mediated by ASIC,P2X, and TRPV1. J Neurophysiol 100:1184–1201.

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