Functional profiling of neurons through
Russell 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 & Research
Academy (SASTRA) University, Tirumalaisamudram, Thanjavur 613401, Tamilnadu, India; andcDepartment of Anesthesiology, University of Utah School of
Medicine, 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, which
should be extendable to neurons in the CNS. In this study, dissoci-
ated dorsal-root ganglion neurons from mice were exposed to
various pharmacological agents (challenge compounds), while at
thesametimethe individualresponses of>100 neurons were simul-
taneously 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 cal-
cium concentration (receptor–agonist challenges) and compounds
that 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 in
different cells (i.e., phenotypes). We used various challenge com-
pounds 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 mem-
brane–potential challenges may be applied to a neuronal popula-
tion to identify, characterize, and discriminate among neuronal
subtypes. This experimental approach can uncover constellations
to 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-focused
neuropharmacology, ultimately leading to the identification and
in the nervous system.
sensory neuron|neuronal subpopulation|conotoxin|conopeptide|Fura-2
identification can be extremely challenging, because at any given
locus in the mammalian nervous system, many functionally di-
verse neurons can be present that are difficult to discriminate
from each other. This lack of cellular differentiation creates
a barrier between systems and molecular neuroscience. Systems
neuroscientists characterize properties of circuits, whereas mo-
lecular neuroscientists identify the signaling macromolecules
that give neurons specific functional properties. Within in-
vertebrate nervous systems, there is a precedent for identifying
different types of neurons morphologically, anatomically, and
physiologically, known as the “identified neuron approach” (1).
Progress in understanding the mammalian nervous system has
been impeded by the lack of a similar paradigm. The gap be-
tween systems and molecular neuroscience in the mammalian
nervous 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 or
neuronal subclass, which may encompass multiple neuronal
subtypes and be identified by the use of a single structural or
or any mechanistic investigation of nervous system function, it
is essential to identify the neurons involved. However, this
functional marker. For example, dorsal-root ganglion (DRG)
neurons may be subdivided into neuronal subclasses defined by
staining with fluorescently labeled isolectin B4(IB4), which binds
to the extracellular matrix proteoglycan, versican, present on the
plasma 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 a
specific neuronal subtype (3–5), particularly at a complex ana-
tomical locus, where potentially hundreds of neuronal subtypes
with 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 been
identified. From a long tradition of anatomical studies pio-
neered by Santiago Ramón y Cajal (6), neurons that have been
most intensively investigated, such as Purkinje and pyramidal
cells, are those cells easily recognized by their striking cell
shapes. However, even morphologically similar pyramidal cells
have been classified into different subclasses based on different
firing properties (7). In most regions of the nervous system, the
vast majority of neurons are not easily differentiated mor-
phologically, further complicating the task of parsing out a
specific neuronal subtype from the surrounding anatomically
Most efforts to differentiate neuronal subtypes broadly use
markers of mRNA or protein expression (3–5, 8–11). Only a few
markers can be used simultaneously, and the expression of
mRNA, or even protein, may not correlate with functional ex-
pression; however, functional expression is the critical parameter
for any mechanistic or physiological study. The principle method
for 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 of
other methods. For this study, we applied pharmacological agents
(challenge compounds) to dissociated mouse lumbar DRG neu-
rons, while monitoring the responses of >100 individual neuronal
cells simultaneously by calcium imaging. Within DRG, >25 sub-
types of neurons are believed to be present based on different
sensory modalities. The divergent responses of individual cells
to 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: firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| January 31, 2012
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distinguishing between neuronal subtypes. The rationale is that
different neuronal subtypes express different receptors and ion
channels in their plasma membranes, which create functional
divergence. The cell bodies of DRG neurons generally are good
surrogates for functional protein expression in axons and nerve
endings 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 have
been used to profile neuronal subtypes in a single experiment
(13–15). We show the feasibility of applying many challenge
compounds in a single experiment, and we have discovered that
certain types of challenge compounds elicited a far greater
spectrum of phenotypic responses than predicted. The successful
application of many challenge compounds, coupled with the
unexpected diversity of response phenotypes, establishes this
experimental 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 our
narrow definition of a neuronal subtype (with a shared, specific
physiological function), because each neuron within the subclass
shares a common structural and functional profile (including
both positive and negative markers) that clearly distinguishes it
from other neuronal subclasses in the DRG cultures.
Preparation and Calcium Imaging of Dissociated Mouse Lumbar DRG
Neurons. Fig. 1 shows bright-field and fluorescence images of
cultured 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 consistent
with previous reports (16). Also shown are ratiometric images
acquired before and during exposure to a high concentration of
potassium (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 an
increase in cytoplasmic calcium concentration, [Ca2+]i, of DRG
neurons. We refer to this experimental strategy as a receptor–
agonist challenge (RA challenge). The second type of protocol
used pharmacological agents targeted to voltage-gated ion
channels, which perturb the membrane potential. A membrane
depolarization that activates voltage-gated Ca channels will
produce an increase in [Ca2+]i. Thus, compounds that act on
voltage-gated ion channels may attenuate or enhance the in-
crease in [Ca2+]ielicited by a membrane depolarization. We
refer to this experimental strategy as a membrane–potential
challenge (MP challenge).
RA Challenge Protocol. With ionotropic receptors (ligand-gated
ion channels), RA challenge compounds directly induce the in-
flux of Ca2+through Ca2+-permeable channels [e.g., transient
receptor potential channel (TRP) V1 receptor] (17). With
metabotropic receptors (G protein-coupled receptors), RA
challenge compounds indirectly elevate cytoplasmic Ca2+by
downstream signaling pathways that ultimately trigger the re-
lease of Ca2+from the endoplasmic reticulum (e.g., histamine
receptors) (18–20). Six different RA challenge compounds were
sequentially 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 experimental
conditions. We refer to experiments using the class of com-
pounds that activate only a minor fraction of the DRG cells as
diagnostic RA challenges.
Definition of DRG Neuron Subclasses Using Diagnostic RA Challenges:
Histamine, ACh, and Menthol.We used diagnostic RA challenges as
a first step to identify a neuronal subtype. DRG neuron sub-
classes that responded to diagnostic RA challenge compounds
could be further subdivided by their responses to other challenge
compounds. We provide a few examples below. One example is
the small percentage of cells that responded to histamine. Ap-
proximately one-half of the histamine-responsive neurons had
the same phenotypic profile: these cells did not respond to the
other diagnostic RA challenge compounds (ACh and menthol)
or AITC but did respond to capsaicin and ATP (Fig. 2B and
Table 2). The average area of the cell soma (cell area) for this
class 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 Table
2). Furthermore, none of those cells stained with Alexa-fluor-
568–labeled IB4 (Fig. S1 and Table 3). A different subclass of
ACh-responsive cells responded to capsaicin and ATP but not to
menthol, histamine, or AITC (Fig. S1 and Table 2), and the
majority 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 cell
area = 670 μm2); in contrast, the subset of ACh-responsive cells
2-AM dye. A–D are images of the same field of view. Fluorescence images
were acquired as described in Materials 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 under
control conditions (i.e., before depolarization). The color scale indicates that
the resting cytoplasmic calcium concentration is relatively low (magenta and
blue). (D) Pseudocolored ratiometric image obtained on depolarization of
the neurons by 100 mM KCl. The color scale, same as the color scale in C,
indicates that the cytoplasmic calcium concentration is relatively high in
many cells (green, yellow, and red). (Scale bar: 30 μm.) Glial cells did not
respond to 100 mM KCl with elevated cytoplasmic calcium, presumably be-
cause they lack voltage-gated calcium channels.
Images of dissociated mouse lumbar DRG neurons loaded with Fura-
Teichert et al.PNAS
| January 31, 2012
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that were also capsaicin- and ATP-sensitive was substantially
smaller (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 also
sensitive to capsaicin, which other investigators have observed
(13, 15, 21–23). A different putative DRG subtype responded
exclusively to menthol (Fig. 2C and Table 2) and did not stain
with IB4 (Table 3). On average, they were exceptionally small
cells (average cell area = 150 μm2) (Table 2) that displayed
unusually noisy and variable resting [Ca2+]i(Fig. 2C). Part of the
variability was characterized by a transient dip in the [Ca2+]i
baseline each time the static bath solution was replaced with
a bath solution containing an RA challenge compound, with the
exception of menthol or KCl, which elicited increases in [Ca2+]i
(Fig. 2C). The size distribution of this class of menthol-sensitive
cells is shown in Fig. 3. Another putative DRG subtype
responded to menthol, AITC, and ATP (Fig. 2C and Table 2)
and stained with IB4 (Table 3). On average, they were larger
cells than those cells that responded only to menthol (average
cell area = 270 μm2) (Table 2).
MP Challenge Protocol. Each neuron has molecular components
with activation that promotes depolarization (e.g., voltage-gated
Na and Ca channels) or hyperpolarization (e.g., voltage-gated K
channels). On application of a depolarizing stimulus, the in-
crease in [Ca2+]iis determined by a balance between the actions
of these two sets of components. Thus, the application of an MP
challenge compound can be used to assess whether particular
voltage-sensitive ion channels are functionally expressed in the
plasma membrane of a neuron by monitoring a decrease or in-
crease in the magnitude and/or kinetics of the calcium signal
elicited 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 challenge
compound, which is described in Materials and Methods and
shown in Fig. 4. To assess how different DRG cells respond to
various MP challenge compounds, we first applied two classical
pharmacological agents: tetrodotoxin (TTX; it inhibits a broad
spectrum of voltage-gated Na channels) and tetraethylammo-
nium (TEA; a standard wide-spectrum, voltage-gated K-channel
blocker). As expected, they produced opposite effects: TTX
decreased 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-induced
calcium signal varied from cell to cell, which is shown in Fig. 5 A–
D. We expected the application of KCl (25 mM) to activate
voltage-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 membrane
sufficiently to activate high-voltage–activated Ca channels.
However, TTX significantly reduced (by >3 SDs below the mean
internal control value) the height of each peak in only a small
subset of cells (<20%), with the high [K+]o-induced calcium
signals of most cells largely nonresponsive to TTX (Fig. 5E).
Diverse Responses to TEA. TEA, at 25 mM, has been shown to
block 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, and
extended the duration of an action potential (25). In view of
these 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.
single neuron’s response to the challenge compounds indicated at the bottom
of 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+]idetermined by the 340/380
nmexcitationratio described inMaterials and Methods. Challenge compounds
were ACh, 1 mM acetylcholine; ATP, 10 μM adenosine 5′-triphosphate; AITC,
menthol; 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 to
the bath.(A–C) Typical sequenceofRAchallenges.Challenge compounds were
applied 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, and
AITC were used to differentiate between neurons only sensitive to menthol
and neurons sensitive toall three challenge compounds,which are highlighted
in blue. (D) Responses from one neuron of each type are shown. Neurons were
also depolarized by KCl (25 mM) pulses at 7-min intervals before and after
application 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 is
for emphasis only. It highlights the different types of responses to TEA.
Example calcium-imaging traces from RA challenges. Each trace is a
| www.pnas.org/cgi/doi/10.1073/pnas.1118833109 Teichert et al.
Some DRG neurons responded directly to TEA addition with
sustained, elevated [Ca2+]iand/or repetitive [Ca2+]ispikes of
variable intermittency (Fig. 5 F and G, respectively). In addition
to these direct effects, many DRG neurons responded to TEA
only with an enhanced response to the subsequent KCl pulse
(Fig. 5 H and I). The enhancement took the form of increased
peak height, width, or both. In all cells, the effects of TEA were
readily reversible. A subset of cells was unresponsive to TEA;
their high [K+]o-induced calcium signals were unaffected by
TEA (Fig. 5J).
Definition of DRG Neuron Subclasses Using Diagnostic MP Challenges:
κM-Conopeptide RIIIJ, Dendrotoxin-K, and Conopeptide pl14a. As
noted above for TEA, K-channel blockers may elicit different
types of phenotypic responses in different cells. Some neurons
responded directly to the challenge compound. This response
typically occurred only in a small proportion of neurons and
could 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 for
KV1.2-containing channels (26), caused a direct response with
sustained, elevated [Ca2+]iin a fraction of large-diameter cells
(Fig. 6). Large-diameter neurons with definitive direct responses
to 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 in
the same subfamily (shaker or KV1). One of these antagonists,
a conopeptide from Conus planorbis (pl14a), is selective for
KV1.6 (27), whereas a kunitz domain small protein from mamba
venom, Dendrotoxin-K (Dtx-K), is reported to be highly selec-
tive for KV1.1 (28). In previous reports, the targeting selectivities
of 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-K
with the direct responses to κM-RIIIJ to evaluate the overlap
between the neurons responsive to pl14a and Dtx-K with the
large-diameter neurons that directly respond to κM-RIIIJ. For
the large-diameter DRG neurons, the response to κM-RIIIJ was
inversely correlated with the response to pl14a (Fig. 6B). For
neurons in which κM-RIIIJ produced robust direct effects, pl14a
produced no direct effects and vice versa. Even more striking was
the fact that those cells that did respond directly to either agent
had different phenotypes. Thus, as illustrated in Fig. 6B, the
smooth, sustained rise in [Ca2+]iobserved with κM-RIIIJ was
strikingly different from the spiky response to pl14a. In contrast,
there was considerable overlap between the cells that responded
directly to Dtx-K and the cells that responded directly to κM-
RIIIJ (Fig. 6C). The direct response of most cells to Dtx-K was
similar 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 responses
constitute another functional phenotype that also subdivides
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, the
toxin’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 a
small, coherent subset of neurons with other correlated pheno-
typic properties. Accordingly, the experiments described above
show that a specific type of phenotypic response to selective
K-channel antagonists can be used as a diagnostic MP challenge.
Additional Characterization of DRG Neuron Subtypes by Combining
RA and MP Challenges. The approach that we used to define
neuronal subclasses also provides opportunities for their further
functional characterization. The menthol-sensitive cells de-
scribed above can be easily identified in the population of DRG
neuronal subtypes. In the experiment shown in Fig. 2D, ATP,
menthol, and AITC were applied to differentiate between two
putative subtypes of menthol-sensitive neurons. The menthol-
sensitive subtype that was insensitive to other RA challenges was
responsive to TEA, an MP challenge; furthermore, in each in-
stance, a characteristic spiky response to TEA was observed. In
contrast, a spiky response to TEA was never observed in the cells
that were sensitive to menthol, ATP, and AITC. Instead, those
cells responded either only indirectly upon high [K+]o-induced
responded to RA challenge compounds
Percentage of mouse lumbar DRG neurons that
Challenge compoundNumber of responsive cellsCells (%)
ACh (1 mM)
ATP (10 μM)
Histamine (50 μM)
Menthol (200 μM)
AITC (mustard oil; 200 μM)
Capsaicin (300 nM)
KCl (100 mM)
Only data from viable neurons, defined by their ability to respond to 100
mM KCl at the end of the trial, are presented in this table and all subse-
Table 2.Selected subclasses of mouse lumbar DRG neurons defined by RA challenges
Diagnostic RA challenge compoundProfiling compoundsDistinctive
*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.
Teichert et al.PNAS
| January 31, 2012
| vol. 109
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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 respond
to other RA challenges were also resistant to all of these MP
challenges, suggesting that inhibition of a different subset of
voltage-gated K channels (non-KV1 family sensitive to TEA) is
responsible for initiating the spiky response to TEA observed in
Fig. 2D. Thus, after a neuronal subtype has been identified by
a set of markers, it may be further characterized by additional
RA and MP challenge compounds.
In Table 3, we summarize the data obtained for seven selected
subclasses of DRG neurons that were defined by diagnostic RA
and/or MP challenges, cell size, and IB4 staining. Some of these
subclasses probably conform to our narrow definition of DRG
subtype because of their highly consistent cross-correlation with
multiple functional and structural markers.
Functional Profiling. This report describes experiments performed
on mouse DRG neurons in which we used a variety of functional
markers to define several distinctive neuronal subclasses. This
analysis has parallels to the early anatomical characterization of
neurons; the classes first recognized by Ramón y Cajal were
those 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 have
chosen to highlight a few neuronal subclasses with clearly dis-
tinctive functional properties. A future objective is to use our
experimental approach, coupled with cluster analysis, to develop
a taxonomy of all neuronal subtypes within the DRG.
Based on cross-correlations of multiple markers, we have
identified 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 as
large-diameter neurons that responded directly to κM-RIIIJ and
medium-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 the
subclasses also showed highly consistent cross-correlations with
several markers (e.g., consistent responses to functional markers
and consistent IB4 staining). Thus, they probably conform to our
narrow definition of neuronal subtypes, with specific common
physiological functions. However, many more DRG subtypes
remain to be identified, and all of the subtypes require addi-
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 functional
phenotypes. Only one subtype responded to AITC and ATP and
stained with IB4, which we will call the MA+ subtype (i.e.
positive responses to menthol, ATP and AITC). The other
subtype was exceptionally small, IB4-negative, and did not re-
spond to any other RA challenge compounds; these cells also
displayed highly variable resting [Ca2+]i(Fig. 2C). We will call
this 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 cellular
neurophamacology. For example, we can investigate the source
of variability in [Ca2+]iobserved in the M+ subtype, which may
be caused by activity of TRPM8 at room temperature. Traces in
Fig. 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
cell size (μm2)
Positive functional markers
(implicated protein expression)
markers IB4 stain
1 Fig. 6 680Direct κM-RIIIJ+* (KV1.2)
2 Figs. 2A, 3A, and 6A 670 Men−, Hist−, Cap−,
Fig. S2480 Indirect κM-RIIIJ+†(KV1.2)IB4+/−
Fig. 3B and S1270 ACh+ (AChRs)
TEA sustained response
TEA spiky response
Men−, Hist−, AITC−
5Fig. 2B 240ACh−, Men−, AITC−
6Fig. 2 C and D270 ACh−, Hist−, Cap−
7Figs. 2 C and D and 3C150 ACh−, Hist−, Cap−,
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-conopeptide
RIIIJ; KV1, KV1 potassium channels; Men, 200 μM menthol; P2X/Y, P2X or P2Y receptors; pl14a, 16 μM conopeptide pl14a; TEA, 10 mM tetraethylammonium
chloride; 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 μm2were included in this percentage. However, the calculation for the average cell area of
480 μm2for subclass 3 included neurons of all diameters that responded indirectly to κM-RIIIJ.
| www.pnas.org/cgi/doi/10.1073/pnas.1118833109Teichert et al.
baseline of the M+ cells each time the static bath solution was
replaced (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 evaporative
cooling of the static bath. Presumably, the warming reduces
TRPM8 activity (i.e., fewer channels open), causing the dip,
followed by evaporative cooling, which increases TRPM8 activity
and thus, restores the [Ca2+]ibaseline after each dip. Some of
the baseline variability may also be related to action potential
bursting 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 isoforms
of various ion channels and receptors present in each menthol-
sensitive subtype. For instance, what voltage-gated Ca-channel
isoforms are present, and do they differ between the M+ and
MA+ subtypes? We can even begin to address which K channels
are present in each subtype and determine whether homomeric
or heteromeric channels are responsible for the functional phe-
notypes observed. The constellation of such receptors and ion
channels in different neuronal subtypes constitute the molecular
basis of their divergent functional properties. Thus, the cellular
neuropharmacology of the menthol-sensitive subtypes should
provide 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 each
neuronal 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 likely
differ. Notably, other investigators have shown that sensory tri-
geminal neurons responsive to innocuous cool temperatures
(low-threshold cold thermoreceptors) show high expression of
TRPM8 and low expression of KV1 channels, whereas neurons
responsive to noxious cold temperatures (high-threshold cold
thermoreceptors) show low expression of TRPM8 and high ex-
pression of KV1 channels, with KV1 acting as an excitability
brake 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), and
instability in [Ca2+]iat room temperature, all consistent with the
hypothesis that the M+ subtype is a low-threshold cold ther-
moreceptor. In contrast, the MA+ subtype typically responded
relatively weakly to menthol (Fig. 2C) (suggesting relatively low
TRPM8 expression), whereas the responses to ATP (31–33) and
AITC are implicated in nociception; the AITC receptor,
TRPA1, is implicated in noxious cold nociception (34–38). These
data, coupled with the relatively stable [Ca2+]iat 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+ and
MA+ neurons in support of these hypotheses, and a detailed
characterization of these two DRG subtypes is presently being
K-Channel Antagonists as Challenge Compounds. In the experiments
described above, a variety of K-channel antagonists were
used. The most extensively analyzed was κM-RIIIJ, reported
to 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 the
subclass 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-
compounds. Data are shown for only a few neuronal subclasses. The x axis is
the same for all panels. Cell area is the area of a cross-section of the cell
soma. (A) ACh only refers to neurons that responded to ACh but not to any
other RA challenge compounds. (B) ACh + ATP + Cap refers to neurons that
responded to ACh, ATP, and capsaicin but no other RA challenge com-
pounds. (C) Menthol only refers to neurons that responded to menthol but
no other RA challenge compound.
Size distributions of neurons responsive to select RA challenge
trace 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 each
KCl pulse (∼15 s) is indicated by the vertical bars (1–8). The first four KCl
pulses (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 monitor
the reversibility of a compound’s effects. The compound of interest was
applied at minute 23 and remained in the bath for 6 min, which is indicated
by 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-elicited
calcium signal, which was readily reversible (indicated by peaks 6–8). (Lower)
Vehicle trials, in which only observation solution was applied to the bath at
minute 23, served as external controls. The red color is for emphasis only. It
highlights the difference between a response to a compound (Upper; com-
pound trial) and a control without a compound (Lower; vehicle trial). At the
end of the experiment, at minute 57, 300 nM capsaicin was applied for 1 min
MP challenge protocol illustrated with calcium-imaging traces. Each
Teichert et al.PNAS
| January 31, 2012
| vol. 109
| no. 5
K, and they were reported to have high selectivity for KV1.6 and
KV1.1, respectively. It is clear that the subset of neurons that
responded 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 that
responded directly to pl14a were, on average, smaller in size.
Thus, different subsets of neurons presumably have inversely
proportional 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 that
responded to κM-RIIIJ and Dtx-K when direct responders were
scored (Fig. 6C). The results suggest that the KV1.1 subunit
overlaps 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 only
slowly reversible in other neurons (Fig. S3), suggesting that Dtx-
K may block different heteromers of KV1.1/1.X with variable
affinity 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 neuropharmacological
approach described in this work. K channels and nicotinic ace-
tylcholine receptors (nAChRs) are examples of ion channels that
endow the nervous system with functional complexity at the level
of individual macromolecules, because the functional receptor/
ion channel is multimeric. Although there is a limited number of
subunit genes, the subunits can combine into heteromers, mak-
ing an enormous array of different combinations possible. For
such ion channel families, it has been challenging to identify the
functional roles for the individual molecular isoforms. The
standard approach to identify function is gene KOs. The prob-
lem for multimeric complexes, such as the K-channel or nAChR
families, is that KO of a single subunit does not just abolish one
isoform but all potential heteromeric combinations containing
the subunit encoded by the KO gene. This problem leads to
a complex phenotype that does not reveal the function of any
individual molecular isoform but rather, is the result of ablating
all 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 the
appropriate neuropharmacological tools (e.g., the α- or κ-con-
opeptides among other reagents), the molecular isoform(s)
challenges. 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 red
color is for emphasis only. It highlights the dif-
ferent types of responses to each challenge
compound. (A–E) Effects of 1 μM TTX, which was
applied for 6 min starting at minute 23 (indicated
by 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 nM
capsaicin was applied for 1 min at minute 57
Selected calcium-imaging traces from MP
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 in
a given panel. Challenge compounds were ACh, 1 mM acetylcholine; C, 300
nM 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 indicate
when each challenge compound or KCl was applied; the latter was applied at
7-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 emphasis
only), after which time the KCl pulses were given before and after applica-
tion of κM-RIIIJ (bar). These neurons were resistant to capsaicin, which is
typical for large-diameter DRG neurons. (B) KCl pulses presented before and
after application of κM-RIIIJ (first bar) and conopeptide pl14a (second bar).
These neurons were resistant to capsaicin. (C) KCl pulses applied before and
after application of κM-RIIIJ (first bar) and Dtx-K (second bar).
Selected traces from large-diameter neurons (cell area > 600 μm2)
| www.pnas.org/cgi/doi/10.1073/pnas.1118833109 Teichert et al.
expressed in specific neuronal subtypes can be identified. There- Download full-text
fore, this identification opens the door to characterize the func-
neuronal circuitry. If one knows the molecular isoform(s)
expressed in a specific neuron, inhibition of each molecular iso-
form should allowan assessment ofthechangein thepropertiesof
the circuit pertaining to that cell. In this way, the cellular neuro-
bridgingtechnology between systems and molecular neuroscience.
Materials and Methods
Mouse DRG Dissection and Cell Culture. Detailed procedures are provided in
SI Materials and Methods. Briefly, WT C57BL/6 mice between the ages of
20–30 d postnatal were euthanized with CO2just before the dissection of
DRGs. Dissection, cell dissociation, and cell culture methods were essentially
as reported previously (39), except the minimal essential media contained
10 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 and
Methods. Briefly, cells loaded with Fura-2 dye were excited intermittently
with 340- and 380-nm light, whereas fluorescence emission was monitored
at 510 nm. An image was captured at each excitation wavelength and the
340/380 nm ratio of fluorescence intensity was acquired (usually) one time
per second to monitor the relative changes in [Ca2+]ifor each cell over time.
Thus, we obtained the 340/380-nm ratiometric images shown in Fig. 1 and
the 340/380-nm ratiometric data (traces) shown in Figs. 2–6. In a given
experiment, >100 cells were individually imaged simultaneously. Increases in
[Ca2+]iwere elicited by ∼15-s application of various RA challenge compounds
or elevated [K+]oin 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 to
avoid issues of dose response. The concentration of ACh was chosen to be
saturating or nearly saturating for all subtypes of ACh receptors. We applied
ATP at the high end of its expected physiological concentration (in muscle
interstitium) (39). The other RA challenge compound concentrations are
consistent with common literature values that have been used to identify
responsive sensory neurons previously (13–15, 19, 35). For MP challenge
compounds, we chose either high concentrations to obtain broad-spectrum
block of K or Na channels (i.e., TEA and TTX, respectively) or relatively low
concentrations that were expected to provide subtype-selective block of
particular 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 were
stained 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+]iduring
the experiment. Such cells were excluded from additional analysis.
ACKNOWLEDGMENTS. We thank Ron Hughen and Robert J. Malcolm for
their advice in the early stages of this work and Lita Imperial for synthesizing
conopeptide pl14a. S.R. acknowledges the Desh Videsh Fund of Shanmugha
Arts, Science, Technology & Research Academy (SASTRA) University. This
work was supported by National Institute of General Medical Sciences
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| vol. 109
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