1Committee on Neurobiology, 2Committee on Cell Physiology, and 3Department of Anesthesia and Critical Care, University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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Genzen, Jonathan R.,
William Van Cleve, and
Daniel S. McGehee.
Dorsal Root Ganglion Neurons Express Multiple Nicotinic
Acetylcholine Receptor Subtypes.
J. Neurophysiol. 86: 1773-1782, 2001.
Although nicotinic agonists can modulate
sensory transmission, particularly nociceptive signaling, remarkably
little is known about the functional expression of nicotinic
acetylcholine receptors (nAChRs) on primary sensory neurons. We have
utilized molecular and electrophysiological techniques to characterize
the functional diversity of nAChR expression on mammalian dorsal root
ganglion (DRG) neurons. RT-PCR analysis of subunit mRNA in DRG tissue
revealed the presence of nAChR subunits 2-7 and
2-
4. Using
whole cell patch-clamp recording and rapid application of nicotinic
agonists, four pharmacologically distinct categories of nicotinic
responses were identified in cultured DRG neurons. Capacitance
measurements were used to divide neurons into populations of large and
small cells, and the prevalence of nicotinic responses was compared between groups. Category I (
7-like) responses were seen in 77% of
large neurons and 32% of small neurons and were antagonized by 10 nM
methyllycaconitine citrate (MLA) or or 50 nM
-bungarotoxin (
-BTX). Category II (
3
4-like) responses were seen in 16% of large neurons and 9% of small neurons and were antagonized by 20 µM
mecamylamine but not 10 nM MLA or 1 µM DH
E. Category II responses
had a higher sensitivity to cytisine than nicotine. Two other types of
responses were identified in a much smaller percentage of neurons and
were classified as either category III (
4
2-like) or category IV
(subtype unknown) responses. Both the
7-like and
3
4-like
responses could be desensitized by prolonged applications of the
analgesic epibatidine.
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INTRODUCTION |
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Dorsal root ganglion (DRG)
neurons convey somatic and visceral sensory information from peripheral
tissues to the spinal cord. DRG neurons are a diverse population, and
different size classes can correlate with modality (Devor
1999). Neurons with small diameter axons and cell bodies are
likely to convey information about pain and temperature, whereas
neurons with larger diameter axons and cell bodies are more likely to
convey mechanoreceptive information. DRG neurons express a diverse
array of neurotransmitter receptors, and this expression can also
correlate with cell size and modality. For example, small-diameter
nociceptive neurons express the vanillinoid receptor (VR-1), which is
sensitive to capsaicin, low pH, and high temperature (Caterina
et al. 1997
; Tominaga et al. 1998
). Interestingly, capsaicin can have both irritative and analgesic effects, presumably due to mechanisms of receptor excitation and desensitization (Fitzgerald 1983
).
In behavioral studies, nicotinic agonists can also induce
analgesic and irritative effects. Receptor knockout experiments have
demonstrated that nicotine-induced analgesia is significantly reduced
in animals that lack the high-affinity 4 and
2-containing nicotinic acetylcholine receptors (Marubio and Changeux 2000
; Marubio et al. 1999
). Although most
reports focus on central sites of action (Damaj et al.
1998
; Davis et al. 1932
; Khan et al.
1994
; Masner 1972
; Sahley and Berntson
1979
), cholinergic agonists excite peripheral sensory nerve
endings (Steen and Reeh 1993
), and there is evidence for
nicotinic acetylcholine receptor (nAChR) expression on both DRG and
trigeminal ganglion (TG) neurons (Boyd et al. 1991
;
Hu and Li 1997
; Liu and Simon 1997
;
Liu et al. 1993
; Morita and Katayama
1989
; Roberts et al. 1995
; Sucher et al.
1990
). There has not been an extensive functional
characterization of the nAChR diversity on mammalian DRG neurons.
Neuronal nAChRs are pentameric ligand-gated ion channels. Nine and
three
subunits have been identified by molecular cloning, and these
subunits assemble in numerous combinations to form functional nAChR
subtypes (Elgoyhen et al. 2001
; Lindstrom
1996
; Lukas et al. 1999
; McGehee
1999
; McGehee and Role 1995
; Sargent
1993
). Receptor subunit composition is the principal
determinant of nAChR properties, including agonist and antagonist
selectivity profiles, as well as the kinetics of activation and
desensitization. Pharmacological characteristics of nAChR-mediated
responses reflect the presence of specific receptor subunits. For
example, the
7 nAChR is activated by millimolar concentrations of
choline and is selectively antagonized by nanomolar concentrations of
methyllycaconitine citrate (MLA) or
-bungarotoxin (
-BTX)
(Alkondon et al. 1992
, 1997
;
Couturier et al. 1990
).
In this study, we utilize a pharmacological approach to assess the prevalence of distinct nAChR subtypes on cultured DRG neurons, focusing on the comparative distribution of nicotinic responses between small and large neurons. Desensitization properties of these responses were also examined.
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METHODS |
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Cell culture
Tissue was isolated from postnatal day 1-3 Sprague-Dawley rats. Briefly, animals were anesthetized with urethan (2 mg/kg ip). DRGs were removed, washed in Hanks' balanced salt solution (HBSS; Life Technologies, Rockville, MD), and dissociated by 60-min trypsin treatment (0.25% in Ca2+-free HBSS) at room temperature followed by mild trituration in modified neuronal MEM (nMEM), which included 10% fetal calf serum (HyClone, Logan, UT), 50 ng/ml 7S NGF (Alomone Labs, Jerusalem, Israel), and 1% Penicillin/Streptomycin (Life Technologies) in Dulbecco's MEM (Life Technologies). nMEM also served as the culture medium. Cells were plated on collagen-coated glass coverslips and maintained at 37° in a humidified incubator with 5% CO2. To limit the problems associated with uncontrolled voltage changes in long neurites, nAChR characterization experiments were carried out using DRG neurons during the first 3 days in culture.
Electrophysiology
Neurons were visualized using an Axiovert 25 inverted microscope
(Zeiss, Göttingen, Germany) and visualized with a ×32 objective with Varel contrast enhancement. Standard external solution (SES) was
constantly perfused at 0.6 ml/min and contained (in mM) 140 NaCl, 2.5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose, pH 7.4. All recordings were done at room
temperature. The pipette solution contained (in mM) 145 K-gluconate, 10 KCl, 1 EGTA, 10 HEPES, 10 glucose, and 5 K-ATP, pH 7.4. Guanosine
triphosphate (100 µM) was added for receptor desensitization
experiments. An EPC7 Patch Clamp (HEKA, Lambrecht, Germany) and a
Digidata 1200 interface (Axon Instruments, Foster City, CA) were used
to digitally acquire data to a Pentium computer running pCLAMP8
software (Axon Instruments). Recording electrodes were manufactured
using a P-97 electrode puller (Sutter Instruments, Novato, CA) and
borosilicate micropipettes (Warner Instruments, Hamden, CT). Electrode
resistance was between 2 and 5 M. Recordings were sampled at 10 kHz
and filtered at 3 kHz. Neurons were identified by their phase-bright appearance, spherical cell bodies, and the presence of
voltage-activated sodium currents as measured in response to
depolarizing voltage steps. Recordings were not included in the
analysis if the series resistance (Rs)
exceeded 25 M
. Rs error correction
was accomplished by postrecording calculation of corrected peak current values.
Drug applications
A piezo-controlled solution exchange system (Piezo Systems; Cambridge, MA) was used for rapid application of nicotinic agonists onto DRG neurons in culture. This system used a three-barreled glass perfusion head (<20 ms exchange at the cell), and cells were exposed to a constant flow of bath solution between drug applications.
Reagents
Nicotine tartrate (Nic), choline chloride (Chol), cytisine
(Cyt), 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), acetylcholine chloride (ACh), methyllycaconitine citrate (MLA), mecamylamine (Mec),
epibatidine (Epi), and dihydro--erythroidine hydrobromide (DH
E)
were all obtained from Sigma/RBI (St. Louis, MO). Capsaicin (Cap) was
obtained from ICN Biochemicals (Aurora, OH).
-Bungarotoxin (
-BTX)
was obtained from Biotoxins (St. Cloud, FL). All drugs were dissolved
in SES. Capsaicin was initially dissolved in 50% ethanol/H2O aliquots at 1,000 times final
concentration, and DMPP was dissolved using a small volume of DMSO at
>1,000 times final concentration. pH and osmolarity were measured for
each drug solution to prevent activation of either proton-sensitive or
mechanoreceptive currents.
RT-PCR
RT-PCR was used to detect mRNA expression in acutely dissected
DRGs. DRGs were obtained using the dissection procedures outlined above. Three DRG equivalents were used per PCR reaction tube. DRGs were
washed in HBSS, which was then subsequently replaced with 1 ml Trizol
reagent (Life Technologies). Tissue was homogenized and chloroform was
added to each tube followed by RNA extraction and precipitation.
Oligo(dT) primers were used to generate cDNA template by reverse
transcription using the GibcoBRL Superscript Preamplification System
(50 min at 42°C). RNase H (Life Technologies) was then used to cleave
the remaining mRNA strand. PCR amplification of specific products was
carried out with the PCR Core System II (Promega, Madison, WI).
Thirty-five PCR cycles were completed with the protocol 95°C 1 min,
55°C 1 min, 73°C 2 min. Products were separated on 2% agarose gel
and visualized with ethidium bromide fluorescence. PCR products were
purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA)
and were subsequently cleaved with appropriate restriction enzymes (New
England Biolabs, Beverly, MA) to verify product length and identity.
Reverse transcriptase-negative controls were run in parallel for each
nAChR subunit assayed to ensure that primers were not amplifying
contaminant DNA. Primer design was conducted using Primer3 Software
(Rozen and Skaletsky 1997), sequence alignments were
conducted using the Wisconsin Package (GCG) with sequences obtained
from GenBank, and restriction maps were generated by DNA Strider
(Marck 1988
). Primers were designed to cross exon
boundaries to distinguish amplified product from mRNA versus genomic
DNA. Primer melting temperatures were determined using the following
equation from Operon, modified for our salt concentrations: Tm = 81.5 + 16.6 × log [Na+] + 41 × (#G + #C)/length
500/length, where [Na+] = 0.075 M. Primer sequences, melting temperature, predicted product
sizes, and restriction enzyme information are included in Table
1.
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Data analysis and statistics
For whole cell currents, Clampfit 8.0 (Axon Instruments) was used to determine current amplitude and rise/decay values. These values were exported to SigmaPlot 5.0 (SPSS, Chicago, IL) for statistical analysis and display. For individual agonist applications, peak amplitude values that were >10 times the baseline RMS noise measured by the Mini Analysis Program (Synaptosoft) were characterized as "responsive."
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RESULTS |
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nAChR mRNA expression in the dorsal root ganglia
We have examined nAChR mRNA expression in the rat DRG using RT-PCR
analysis. Ganglia were removed from neonatal pups of the same age used
for our primary cultures (1-3 days postnatal), and mRNA was isolated
from a combined pool of excised DRGs from both male and female pups.
PCR primer information is located in Table 1. As Fig.
1A indicates, amplification
products of the predicted sizes were detected for each of the alpha
(2,
3,
4,
5,
6, and
7) and beta subunits (
2,
3,
and
4) examined. The chick
8 (Anand and Lindstrom
1992
) and vestibulocochlear
9 (Elgoyhen et al.
1994
) were not examined.
2 expression was faint but visually detectable. In all cases, reverse transcriptase-negative controls yielded no product, ensuring that amplified template was originally from mRNA sources. Figure 1B illustrates the results of
restriction enzyme treatment of the PCR products, which yielded
fragments of the predicted sizes as indicated in Table 1.
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DRG neuron size categorization
Electrophysiological recordings were made from neonatal DRG neurons in culture. Membrane capacitance measurements were recorded for every neuron in our experiments and were used to divide cells into distinct size categories. Cell diameter measurements were obtained for a subpopulation of neurons using a calibrated reticle and were calculated as an average of the shortest and longest axes of each soma. A plot comparing capacitance and diameter measurements for a representative sample of DRG neurons is shown in Fig. 2. The solid line represents the relationship between membrane capacitance and diameter of a spherical cell, based on the specific membrane capacitance of 1 µF/cm2. Divergence of data points from this line is likely the result of increased membrane area of the neuronal processes. A threshold of 18.7 pF was used as the cutoff between large and small neurons (dashed line). The mean ± SD of capacitance measurements from 86 visually identified small neurons is 18.7 pF (average diameter, 16.0 ± 2.7 µm, mean ± SD).
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DRG neurons respond to nicotinic agonists
A rapid, piezo-controlled solution exchange system was used for nicotinic agonist applications onto DRG neurons in culture (Fig. 3A). Nicotinic agonists evoked rapid excitatory currents in a concentration-dependent manner in some DRG neurons (Fig. 3B). Initial experiments determined appropriate agonist concentration ranges (between 50 and 80% of the maximal response), which were then utilized for the more extensive pharmacological characterization below. Currents were observed following application of several nicotinic agonists including epibatidine, DMPP, cytisine, nicotine, acetylcholine, and choline.
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Category I response: functional evidence for 7 receptor
expression by DRG neurons
The initial experiments indicated that a subpopulation of DRG
neurons responded to choline. As choline has been reported to act as an
7-selective agonist (Alkondon et al. 1997
), we tested the nicotinic responses in choline-sensitive neurons pharmacologically to determine whether DRG neurons express functional
7 receptors. Figure 4A shows a DRG neuron
that responds to both choline (10 mM) and acetylcholine (1 mM). Both
the choline and acetylcholine-induced currents in this cell were
reversibly blocked by a 3-min preincubation with the
7-selective
antagonist MLA (10 nM; Fig. 4B). After a 12-min wash, both
agonists evoked rapidly activating currents again. The choline and
acetylcholine-induced currents were then completely blocked by an
irreversible
7 antagonist,
-BTX (50 nM; Fig. 4C). No
current could be activated by either choline or acetylcholine even
after a 40-min wash, which is consistent with the
7 subtype nAChR.
In five of five cells tested with both antagonists, choline-induced
currents were completely blocked by 10 nM MLA and 50 nM
-BTX.
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Prevalence, amplitude, rise time, and decay information for each of the
nicotinic response categories is presented in Table 2. A total of 220 neurons was surveyed
for the presence of functional 7 receptors. The
7 receptors were
the most common nAChR in DRG neurons. Seventy-seven percent of large
neurons and 32% of small neurons had these category I responses, which
had rapid activation and desensitization kinetics. There were no
statistical differences in current density, rise time, or decay time
for category I currents between the populations of large and small DRG
neurons.
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We compared agonist potency on 7-expressing DRG neurons by testing
responses to 100-µM test doses (n = 3 cells per
agonist) and then normalizing to the nicotine response (Fig.
4D). Neurons were included in these rank order potency
experiments if the responses to both choline and ACh were completely
blocked by 10 nM MLA (as in Fig. 4, A and B).
Although choline is the least potent agonist at
7 receptors, it is
also selective, even at high millimolar concentrations. The rank order
potency for nicotinic agonists at
7-expressing neurons was as
follows: DMPP
cytisine > nicotine
acetylcholine > choline.
Category II response: functional evidence for 3
4 receptor
expression by DRG neurons
While conducting the pharmacological characterization of 7
responses in DRG neurons, MLA-insensitive ACh currents were also observed in some cells. These MLA-insensitive currents could also be
activated by cytisine, which is a full agonist at
4-containing receptors yet only a partial agonist at receptors containing the
2
subunit (Papke and Heinemann 1994
). Figure
5A shows an example of a
cytisine-induced current that was not blocked by 10 nM MLA. To examine
the properties of these non-
7 currents, 10 nM MLA was included in
perfusion solutions.
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A total of 266 DRG neurons was surveyed for the expression of
MLA-insensitive nAChRs. Under these conditions, neurons that responded
to cytisine always displayed a greater sensitivity to cytisine than
nicotine, regardless of the order of application. For example, Fig.
5B shows a cytisine-induced current in the presence of 10 nM
MLA compared with a nicotine-induced current in the same cell. These
category II responses were the second-most common nicotinic response in
DRG neurons, seen in 16% of large cells and 9% of small cells (see
Table 2). Category II responses also had slower activation and
desensitization kinetics than were observed for 7. There were no
statistical differences in current density, rise time, or decay time
for category II currents between the populations of large and small DRG neurons.
The rank order potency of nicotinic agonists on category II responses
[using 50-µM test doses for all drugs except epibatidine (1 µM);
n 3 cells per agonist] was as follows: epibatidine
acetylcholine
cytisine
DMPP > nicotine
choline (Fig.
5C). These MLA-insensitive currents could be blocked by 20 µM mecamylamine (Fig. 5D; n = 6) but not 1 µM DH
E (n = 3). The cell shown in Fig.
5D was also sensitive to 10 µM capsaicin (data not shown). Category II responses were therefore characterized by insensitivity to
10 nM MLA or 1 µM DH
E, activation by cytisine with a greater potency than nicotine, and antagonism by 20 µM mecamylamine.
These observations point to the expression of
3
4 nAChRs in a
subpopulation of DRG neurons (see DISCUSSION for additional evidence).
Category III and IV responses: functional evidence for two other categories of nicotinic responses in DRG neurons
While investigating the properties of category II responses on DRG neurons, two other categories of responses were also observed. These responses were identified in a much smaller population of neurons, which precluded extensive pharmacological characterization.
Of the neurons used for the characterization of category II
responses, a category III (4
2-like) response was seen in 4% the
large DRG neurons and in <1% of the small DRG neurons (see Table 2).
This category III response was insensitive to cytisine (50 µM;
n = 6), but activated by nicotine (n = 2), DMPP (n = 1), or epibatidine (n = 3). These currents could be antagonized by DH
E (100 nM,
n = 1; 1 µM, n = 2), a potent
selective antagonist of
4
2-receptors (Alkondon and
Albuquerque 1993
; Luetje et al. 1990
). Figure
6A shows representative
currents from a small DRG neuron that was insensitive to 50 µM
cytisine but activated by 50 µM nicotine. This current could be
reversibly antagonized by 100 nM DH
E. The profound insensitivity to
cytisine indicates that this receptor subtype is not likely to contain
the
4 subunit, and antagonism by DH
E in nanomolar concentrations
serves as evidence of an
4
2-like nAChR subtype in a small
fraction of DRG neurons in culture.
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One final category of response was observed in DRG neurons. These
category IV responses were relatively large compared with those
described above, were insensitive to cytisine, and had much slower
activation and desensitization kinetics (see Table 2). Figure
6B shows one large DRG neuron in which 25 µM nicotine
induced a response that lasted over 100 s with a persistent
current of >1 nA. When we reviewed all of the recordings from these
experiments (including preliminary analyses and receptor
categorizations I-III), category IV responses were evident in 3% of
large neurons and in 1% of small neurons. These slower responses have
been observed with nicotine (n = 6), epibatidine
(n = 4), and choline (n = 4). Category
IV responses could also be discriminated from categories I-III by
their insensitivity to 10 nM MLA (n = 5), 100 nM
-BTX (n = 3), 1 µM DH
E (n = 2),
and 100 µM mecamylamine (n = 1).
Functional consequences of nAChR expression on sensory neurons
This pharmacological investigation illustrates the diversity of nAChR subtypes expressed by DRG neurons. As nAChR desensitization might interfere with any endogenous cholinergic transmission, we have examined the desensitization properties of these nicotinic responses in DRG neurons.
As is shown in Fig. 7A, a DRG
neuron that initially responds to a 100-µM ACh application with an
inward current no longer responds after the receptors are desensitized
by a prolonged epibatidine application. The kinetics of recovery from
desensitization were investigated for both category I and II responses
in DRG neurons and are shown in Fig. 7B. Epibatidine
applications (1 µM, 30 s) were used to desensitize responses to
100 µM ACh, and recovery from desensitization was measured using
subsequent ACh applications every 40-60 s. The data were normalized to
an initial 100-µM ACh response before desensitization and was
corrected for measured rundown observed in the absence of epibatidine
desensitization. Recovery times from desensitization were remarkably
similar for category I and II responses ( of 243 and 234 s,
respectively), although the initial rate of desensitization for these
categories is different (see Table 2). If endogenous cholinergic
transmission plays a role in sensory transduction or transmission,
desensitization of nAChRs by exogenous nicotinic agonists could inhibit
this endogenous activity.
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DISCUSSION |
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The results of this study show that DRG neurons express multiple
subtypes of nAChRs, and that the distribution of receptor expression
varies according to cell size. RT-PCR analysis of mRNA expression in
DRG tissue indicated the presence of nearly every known nAChR subunit
(2-7,
2-4). A pharmacological approach was chosen to detect and
characterize the nAChRs functionally expressed on DRG neurons in
culture, and four response categories were observed.
Category I (7-like) responses were seen in 77% of large neurons and
in 32% of small neurons. These findings demonstrate that the
7
nAChR subtype is the most prevalent nAChR expressed by neonatal DRG
neurons in culture. The exact role of
7 receptors on these neurons
is unclear, although
7 receptors are known to play a role in
presynaptic modulation of glutamatergic transmission in other systems
(McGehee et al. 1995
), and we have preliminary evidence
indicating that this also occurs in the dorsal horn of the spinal cord,
the central projection site of DRG neurons (Genzen and McGehee
1999
). The
7 receptor is unique in that the channel is
extremely permeable to Ca2+ (Seguela et
al. 1993
), which makes it an ideal channel for presynaptic modulation, or as an activator of Ca2+-mediated
second-messenger cascades. This report demonstrates functional
7
receptors on primary somatosensory neurons, and it provides an
explanation for the autoradiographic evidence for
-BTX binding sites
on sensory neurons demonstrated two decades ago (Ninkovic and
Hunt 1983
; Polz-Tejera et al. 1980
).
Additionally, intense
7 immunolabeling has been detected in the
spinal mesencephalic nucleus of the trigeminal nerve, a region known to
contain jaw muscle afferents (Dominguez del Toro et al.
1994
).
Category II (3
4-like) responses were the second most common
response observed in DRG neurons, seen in approximately 16% of large
neurons and in 9% of small neurons. The
3
4 designation of
category II responses is based on the similarity between the pharmacological profile we observed and that of nAChRs generated by
pair-wise expression in Xenopus oocytes. This idea is also supported by immunoprecipitation and binding experiments in sensory neurons. The relative potency of cytisine versus nicotine can be used
to implicate the presence of the
4 subunit (Lena et al. 1999
), which is the case for our category II response. While we observed a higher sensitivity to ACh and DMPP than was seen in oocyte
expression studies, the similar potency of these two agonists is
consistent with
3
4 expression (Luetje and Patrick
1991
; Papke and Heinemann 1994
). Evidence for
3
4 receptor expression has also been demonstrated in trigeminal
ganglion tissue (Flores et al. 1996
). The prevalence of
category II responses in our experiments matched the distribution of
3 mRNA transcripts seen using in situ hybridization in both chick
DRG and rat trigeminal ganglion (Boyd et al. 1991
;
Flores et al. 1996
).
The less common category III response (4
2-like) was seen in
approximately 4% of large neurons and <1% of small neurons. These
responses were remarkably insensitive to cytisine and were antagonized
by DH
E. Our results therefore suggest that functional
4
2
nAChRs are expressed in only a small percentage of DRG neurons, although it is unknown whether this expression is rare in all classes
of DRG neurons, or possibly restricted to a modality-specific subpopulation. The low prevalence of category III responses in our
culture preparation closely matched the distribution of
4 mRNA
transcripts in chick DRGs (Boyd et al. 1991
), although
less so with the distribution in adult rat trigeminal ganglion
(Flores et al. 1996
), where a more diffuse labeling was
seen in up to 60-80% of neurons.
Category IV responses remain unclassified due to rare expression in our culture conditions, although the strikingly different time course, amplitude, and pharmacology are worthy of note. Category IV responses were seen in 3% of large neurons and 1% of small neurons and could be activated by nicotine, epibatidine, or choline but not cytisine.
It should be noted that other nAChR subunits are likely to be included
in each of the receptor categorizations above. 5,
6, and
3
subunit mRNA expression was also detected in DRG tissue by our RT-PCR
analysis, and these subunits have been shown to co-assemble with other
nAChRs to create more diverse receptor profiles. For example,
5
addition to
3
4 or
3
2 receptors expressed in
Xenopus oocytes enhances receptor desensitization and
increases Ca2+ permeability (Gerzanich et
al. 1998
).
6 and
3 might also contribute to functional
nAChRs. These subunits are largely colocalized in the CNS (Le
Novere et al. 1996
), and mRNAs for both subunits are coexpressed in a subpopulation of nicotine-responsive locus coeruleus neurons (Lena et al. 1999
). We do not know whether these
subunits contribute to the category IV response, or whether this
response is due to an unidentified receptor protein complex. The
suggested receptor subunit compositions outlined above are likely to
represent minimal receptor complexes that may in fact contain
additional nAChR subunits (Lukas et al. 1999
).
While the present results are derived from agonist applications onto
the somata of cultured DRG neurons, it is reasonable to question
whether nAChRs are also expressed on the terminals of DRG afferents.
Evidence for peripheral nAChR expression comes from single fiber nerve
recordings by Steen and Reeh (1993), who demonstrated
that nicotinic activation of peripheral nerve terminals can result in
excitation. Single-unit spinal cord microelectrode recordings have also
demonstrated that most wide-dynamic range dorsal horn neurons respond
to intracutaneous nicotine application (Jinks and Carstens
1999
), and nicotinic agonists have also been shown to excite
afferents in the rabbit cornea (Tanelian 1991
). Furthermore, Ninkovic and Hunt (1983)
observed evidence
for both peripheral and central and transport of
-BTX binding sites
on sensory neurons, and dorsal horn
-BTX binding is largely
eliminated after dorsal rhizotomy.
The spinal cord contains a subpopulation of inhibitory interneurons
that possess the synthesizing enzyme for acetylcholine (ChAT, choline
acetyltransferase), and ChAT-positive varicosities can make presynaptic
contact onto to the terminals of primary afferents in the dorsal horn
(Barber et al. 1984; Ribeiro-da-Silva and Cuello
1990
; Todd 1991
). Acetylcholine in the spinal
cord might therefore activate nAChRs expressed on the central terminals of DRG afferents. It is also possible that choline, a product of ACh
degradation by acetylcholinesterase, may also activate these receptors
depending on concentrations at the synapse. The sources of
acetylcholine that could activate nAChRs on peripheral nerve terminals
are much more speculative, although there is evidence that many
nonneuronal cells either contain or can manufacture acetylcholine
(Wessler et al. 1999
).
While peripheral applications of nicotinic agonists can cause the
excitatory effects described above, behavioral experiments have
demonstrated that nicotinic agonists can also have analgesic properties
when delivered via multiple routes of administration (Davis et
al. 1932; Masner 1972
; Sahley and
Berntson 1979
). Epibatidine is a nicotinic agonist with an
analgesic potency greater than morphine (Spande et al.
1992
), and ABT-594 (a novel nicotinic agonist synthesized by
Abbott Laboratories) can produce analgesia when administered
systemically, intradermally, or centrally in the nucleus raphe magnus,
a site involved in the descending modulatory system of analgesia
(Bannon et al. 1998
; Bitner et al. 1998
;
Boyce et al. 2000
; Kesingland et al.
2000
). While some reports have identified dose-dependent
analgesia after both intrathecal and subcutaneous applications of
nicotinic agonsts (Damaj et al. 1998
), others
demonstrate irritative and autonomic responses (Khan et al.
1994
) or hyperalgesia at subanalgesic doses (Masner
1972
). It is likely that the variation in response to nicotinic
agonist administration in vivo may involve a number of factors, and
identification of nAChR properties on sensory neurons in vitro will
help reveal the underlying mechanisms.
Excitatory effects of nicotinic agonists are primarily due to nAChR
activation, and subsequent neuronal depolarization leads to
Na+ or Ca2+-channel
activation. Inhibitory effects may be due to receptor desensitization,
Na+ or Ca2+-channel
inactivation, Ca2+-mediated second-messenger
pathways, or simply current shunting. Of the inhibitory effects,
receptor desensitization occurs at much lower agonist concentrations
than those needed for activation (Fenster et al. 1997)
and may also play an important role in modulating sensory activity.
Discrepancies between analgesic, hyperalgesic, or irritative actions of
nicotinic ligands might therefore be due to a complex balance between
receptor expression patterns and basic drug pharmacokinetics and
pharmacodynamics. The fact that a majority of large DRG neurons express
functional nAChR implies that modulation of nonnociceptive modalities
could contribute to the behavioral effects of these compounds. For
example, subpopulations of visceral sensory afferents in the nodose
ganglia respond to cholinergic agonists (Baccaglini and Cooper
1982) and are labeled by
-BTX autoradiography
(Ashworth-Preece et al. 1998
), supporting the idea that
nAChRs may play a role in the modulation of visceral as well as somatic
sensory transmission. Understanding the sensory effects of nicotinic
agonists will necessarily involve identifying the specific nAChRs
expressed at each location along these sensory pathways.
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ACKNOWLEDGMENTS |
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The authors thank Drs. Amy MacDermott and Huibert Mansvelder for comments on the manuscript.
This work was supported by National Institutes of Health Grants NS-35090 (D. S. McGehee) and 1F30DA-06033 (J. R. Genzen), the Growth and Development Training Program T32HD-07009 (J. R. Genzen), and the Brain Research Foundation.
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FOOTNOTES |
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Address for reprint requests: D. S. McGehee, University of Chicago, Dept. of Anesthesia and Critical Care, 5841 S. Maryland Ave. MC4028, Chicago, IL 60637 (E-mail: dmcgehee{at}midway.uchicago.edu).
Received 6 April 2001; accepted in final form 2 July 2001.
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REFERENCES |
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