Expressing Primary Afferents by [D-Ala2,N-Me-Phe4,Gly-ol5...

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Sustained Inhibition of Neurotransmitter Release from non-TRPV1-

Expressing Primary Afferents by [D-Ala2,N-Me-Phe4,Gly-ol5]-Enkephalin in

the Spinal Cord

Hong-Yi Zhou, Shao-Rui Chen, Hong Chen, and Hui-Lin Pan

Department of Anesthesiology and Pain Medicine (HYZ, SRC, HC, HLP)

The University of Texas M. D. Anderson Cancer Center

Houston, TX 77030

and

Program in Neuroscience (HLP)

The University of Texas Graduate School of Biomedical Sciences

Houston, TX 77225

JPET Fast Forward. Published on July 31, 2008 as DOI:10.1124/jpet.108.141226

Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on July 31, 2008 as DOI: 10.1124/jpet.108.141226

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Running title: Primary afferent phenotypes and µ-opioid receptors List of Abbreviations:

Griffonia simplicifolia isolectin B4 (IB4); [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO);

transient receptor potential vanilloid type 1 (TRPV1); excitatory postsynaptic currents (EPSCs);

miniature excitatory postsynaptic currents (mEPSCs); G protein-coupled inwardly rectifying K+

channels (GIRK)

Number of text pages: 33;

Number of tables: 0;

Number of figures: 8;

Abstract, 244;

Introduction, 454;

Discussion, 1413;

Number of references: 32

Address for correspondence: Hui-Lin Pan, M.D., Ph.D. Department of Anesthesiology and Pain Medicine, Unit 110 The University of Texas M. D. Anderson Cancer Center 1515 Holcombe Blvd. Houston, TX 77030 Tel: (713) 563-5838 Fax: (713) 794-4590 email: [email protected]


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Abstract

Removing transient receptor potential vanilloid type 1 (TRPV1)-expressing primary

afferent neurons reduces presynaptic µ-opioid receptors but potentiates opioid analgesia.

However, the sites and underlying cellular mechanisms for this paradoxical effect remain

uncertain. In this study, we determined the presynaptic and postsynaptic effects of the µ-opioid

receptor agonist (D-Ala2,N-Me-Phe4,Gly-ol5)-enkephalin (DAMGO) using whole-cell patch-

clamp recordings of lamina II neurons in rat spinal cord slices. Treatment with the ultrapotent

TRPV1 agonist resiniferotoxin (RTX) eliminated TRPV1-expressing DRG neurons and their

central terminals in the spinal dorsal horn and significantly reduced the basal amplitude of

glutamatergic excitatory postsynaptic currents (EPSCs) evoked from primary afferents.

Although RTX treatment did not significantly alter the concentration-response effect of

DAMGO on evoked monosynaptic and polysynaptic EPSCs, it causes a profound long-lasting

inhibitory effect DAMGO on evoked EPSCs. Subsequent naloxone treatment did not reverse

the prolonged inhibitory effect of DAMGO on evoked EPSCs. Furthermore, brief application of

DAMGO produced a sustained inhibition of miniature EPSCs in RTX-treated rats. However,

the concentration-response and the duration of DAMGO’s effect on G protein-coupled inwardly

rectifying K+ currents in lamina II neurons were not significantly different between vehicle- and

RTX-treated groups. These data suggest that stimulation of µ-opioid receptors on non-TRPV1

afferent terminals causes extended inhibition of neurotransmitter release to spinal dorsal horn

neurons. The differential effect of µ-opioid receptor agonists on different phenotypes of primary

afferents provides a cellular basis to explain why the analgesic action of opioids on mechano-

nociception is prolonged when TRPV1-expressing primary afferents are removed.


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Introduction

The primary afferent neurons and the spinal cord are critically involved in pain

transmission and modulation and are the important sites for the analgesic action of µ-opioid

receptor agonists (Yaksh and Noueihed, 1985; Magnuson and Dickenson, 1991; Kohno et al.,

1999; Chen and Pan, 2006a). The µ-opioid receptors are located presynaptically on primary

afferent terminals (Fields et al., 1980; Kohno et al., 1999; Abbadie et al., 2002) and on

postsynaptic dorsal horn neurons (Light and Willcockson, 1999; Marker et al., 2006).

Presynaptically, the µ-opioid receptor agonists reduce excitatory neurotransmitter release

through presynaptic inhibition of voltage-gated Ca2+ channels (Rusin and Moises, 1995; Wu et

al., 2004) and direct inhibition of synaptic vesicle releases (Blackmer et al., 2005; Gerachshenko

et al., 2005). However, the contributions of µ-opioid receptors distributed on different

phenotypes of primary afferent terminals to the opioid analgesia are not clear.

The capsaicin receptor transient receptor potential vanilloid type 1 (TRPV1) plays an

essential role in detecting thermal nociception (Caterina et al., 2000). Furthermore, removing

TRPV1-expressing dorsal root ganglion (DRG) neurons by resiniferatoxin (RTX), an ultrapotent

TRPV1 agonist, impairs thermal nociception in adult rats (Chen and Pan, 2006b). It has been

shown that TRPV1 antagonists reduce mechanical nociception in acute and chronic pain models

(Walker et al., 2003; Cui et al., 2006). Interestingly, although RTX induces a substantial

reduction in presynaptic µ-opioid receptors, it potentiates and prolongs the analgesic effect

produced by systemic or intrathecal injection of µ-opioid receptor agonists, including morphine

and DAMGO (Chen and Pan, 2006b). Removal of TRPV1-expressing sensory neurons by RTX


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also attenuates the development of morphine analgesic tolerance (Chen et al., 2007). However,

the cellular and molecular mechanisms underlying this paradoxical opioid action in RTX-treated

rats are unclear. The phenotypes and functions of DRG neurons, even among the small-diameter

neurons, are highly heterogeneous. It has been shown that the µ-opioid agonists have a greater

effect on voltage-gated Ca2+ channels in IB4-negative small-sized DRG neurons than in IB4-

positive (mostly TRPV1-expressing) ones (Wu et al., 2004). Furthermore, the µ-opioid receptor

agonists such as morphine reduce the mechanical nociception far longer than thermal

nociception (Chen and Pan, 2006a). The potential difference in the functional activity of µ-

opioid receptors on TRPV1- and non-TRPV1-expressing afferent terminals has not been

determined previously.

The aim of the present study, therefore, was to determine whether removing TRPV1-

expressing sensory neurons alters the presynaptic and postsynaptic effects of the µ-opioid

receptor agonist in the spinal cord. Our findings provide new evidence that loss of TRPV1-

expressing sensory neurons causes the µ-opioid receptor agonist to have a profound, long-lasting

presynaptic, but not postsynaptic, effect in the spinal dorsal horn. This information is important

for our understanding of the functions of the µ-opioid receptors present in different phenotypes

of primary afferent neurons.


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Methods

Animals and RTX treatment

Male Sprague-Dawley rats (130-150 g; Harlan, Indianapolis, IN) were used in this study.

Rats received a single intraperitoneal injection of RTX (200 µg/kg, LC Laboratories, Woburn,

MA) while under 2-3% isoflurane anesthesia, and the anesthesia was maintained for 3 h after

RTX injection (Chen and Pan, 2006b). RTX was dissolved in a mixture of 10% Tween-80 and

10% ethanol in normal saline. Rats in the control group received intraperitoneal injection of

vehicle alone. The final electrophysiological experiments were conducted 4 weeks after RTX

and vehicle injections. All the surgical preparation and experimental protocols were approved

by the Animal Care and Use Committee of the University of Texas M. D. Anderson Cancer

Center and conformed to the National Institutes of Health guidelines for the ethical use of

animals.

Behavioral assessment of thermal nociception

To quantitatively assess the thermal nociceptive threshold, rats were placed on the glass

surface of a thermal testing apparatus (IITC Inc., Woodland Hills, CA). The rats were allowed to

acclimate for 30 min before testing. The temperature of the glass surface was maintained

constant at 30EC. A mobile radiant heat source located under the glass was focused onto the

hindpaw. The paw withdrawal latency was recorded using a timer. A cutoff of 30 s was used to

prevent potential tissue damage (Chen and Pan, 2006a).


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Double fluorescence labeling of TRPV1 and IB4 in DRGs and spinal dorsal horn

To ensure the effect of RTX treatment on TRPV1-expressing DRG neurons and

terminals, double labeling of TRPV1 and Griffonia simplicifolia isolectin B4 (IB4, a cellular

marker for unmyelinated afferent fibers) in DRG neurons and the spinal dorsal horn was

performed on three vehicle- and three RTX-treated rats 4 weeks after treatment (Chen and Pan,

2006b). Under deep anesthesia (pentobarbital sodium, 60 mg/kg, ip), each rat was intracardially

perfused with 4% paraformaldehyde and 10% sucrose. The lumbar spinal cord and L4 and L5

DRGs were removed and postfixed in the same fixative solution and cryoprotected in 30%

sucrose in PBS for 48 h at 4EC. The tissues were cut to 30 µm in thickness and collected free-

floating in 0.1 M PBS. For TRPV1 labeling, the sections were first blocked in 4% normal goat

serum in PBS for 1 h. The sections were then incubated with the primary antibody (rabbit anti-

TRPV1 N-terminus, dilution 1:1000, Neuromics, Minneapolis, MN) diluted in PBS containing

2% normal goat serum and 0.3% TX-100 for 2 h at room temperature and overnight at 4EC.

Subsequently, sections were incubated with the secondary antibody (Alexa Fluor-488 conjugated

to goat anti-rabbit IgG, dilution: 5 µg/ml, Molecular Probes, Eugene, OR). The sections then

were rinsed in PBS and incubated with Alexa Fluor-594 conjugated to IB4 (dilution: 2 µg/ml,

Molecular Probes) for 2 h at room temperature. Finally, the sections were mounted on slides,

dried, and coverslipped. The sections were examined on a laser scanning confocal microscope

(Zeiss, Germany), and areas of interest were photodocumented. Omission of the primary

antibody results in negative labeling in all the sections examined.

Spinal cord slice preparation


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With the rats under 2-3% isoflurane anesthesia, we removed the lumbar segment of the

spinal cord by means of laminectomy. This spinal cord segment was immediately placed in an

ice-cold sucrose artificial cerebrospinal fluid (aCSF) presaturated with 95% O2 and 5% CO2.

The sucrose aCSF contained (in mM) 234 sucrose, 3.6 KCl, 1.2 MgCl2, 2.5 CaCl2, 1.2

NaH2PO4, 12.0 glucose, and 25.0 NaHCO3. The tissue was then placed in a shallow groove

formed in a gelatin block and glued on the stage of a vibratome (Technical Product

International, St. Louis, MO). Transverse spinal cord slices (400 µm) were cut in the ice-cold

sucrose aCSF and then preincubated in Krebs solution oxygenated with 95% O2 and 5% CO2 at

34EC for at least 1 hour before being transferred to the recording chamber. The Krebs solution

contained (in mM) 117.0 NaCl, 3.6 KCl, 1.2 MgCl2, 2.5 CaCl2, 1.2 NaH2PO4, 11.0 glucose, and

25.0 NaHCO3.

Electrophysiological recordings

Postsynaptic currents were recorded using the whole-cell voltage-clamp method, as we

described previously (Zhang et al., 2007; Zhou et al., 2007). The slice was placed in a glass-

bottomed chamber (Warner Instrument, Hamden, CT) and fixed with parallel nylon threads

supported by an U-shaped stainless steel weight. The slice was continuously perfused with

Krebs solution at 5.0 ml/min at 34EC maintained by an inline solution heater and a temperature

controller (TC-324; Warner Instrument). The lamina II was identified by light microscopy as a

distinct translucent band across the superficial dorsal horn. The neurons in the lamina II of the

spinal cord slice were identified by fixed-stage microscopy (BX50WI; Olympus, Tokyo, Japan)

with differential interference contrast/infrared illumination. The spinal lamina II neurons were


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studied because they receive predominantly nociceptive input from both TRPV1- and non-

TRPV1-expressing C-fiber afferent terminals (Woodbury et al., 2000; Pan et al., 2003).

Monosynaptic and polysynaptic excitatory postsynaptic currents (EPSCs) were evoked

by electrical stimulation (0.6 mA, 0.2 ms) of the dorsal root or dorsal root entry zone (Zhang et

al., 2007). The electrode for the whole-cell recordings was pulled from borosilicate glass

capillaries with a puller (P-97; Sutter Instrument Company, Novato, CA). The impedance of the

pipette was 3-5 MΩ when filled with internal solution containing (in mM) 110 Cs2SO4, 5 TEA,

2.0 MgCl2, 0.5 CaCl2, 5.0 HEPES, 5.0 EGTA, 5.0 ATP-Mg, 0.5 Na-GTP, 1 guanosine 5'-O-(2-

thiodiphosphate) (GDP-β-S), and 10 lidocaine N-ethyl bromide (QX314) that had been adjusted

to pH 7.2 to 7.4 with 1 M CsOH (290-300 mΩsm). To examine the presynaptic effect of the µ-

opioid receptor agonist (D-Ala2,N-Me-Phe4,Gly-ol5)-enkephalin (DAMGO), GDP-β-S and K+

channel blockers (Cs+ and TEA) were added to the internal solution to inhibit the postsynaptic

effect of DAMGO. QX314 was added into the internal solution to suppress the action potential

generation from the recorded cell. The evoked EPSCs were considered to be monosynaptic if: 1)

the latency was constant with repeated electrical stimulation at 0.1 Hz, and 2) there was no

conduction failure or increased latency when stimulation frequency was increased to 20 Hz

(Zhang et al., 2007). In contrast, evoked EPSCs were considered polysynaptic if the latency was

variable and conduction failure occurred at the higher stimulation frequency (20 Hz). To record

the miniature EPSCs (mEPSCs), 1 µM tetrodotoxin (TTX) was added to the perfusion solution.

To study the postsynaptic effect of the opioid agonist, G protein-coupled inwardly

rectifying potassium (GIRK) currents induced by DAMGO were recorded in lamina II neurons

(Marker et al., 2006). The impedance of the pipette was 5-10 MΩ when filled with internal


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solution containing (in mM) 135.0 potassium gluconate, 5.0 TEA, 2.0 MgCl2, 0.5 CaCl2, 5.0

HEPES, 5.0 EGTA, 5.0 ATP-Mg, 0.5 Na-GTP; the solution was adjusted to pH 7.2 to 7.4 with 1

M KOH (290-300 mΩsm). GDP-β-S and QX-314 were not included in the intracellular solution

for the recording of GIRK currents.

Recordings of evoked EPSCs, mEPSCs, and GIRK currents began about 6 min after

whole-cell access was established and the current reached a steady state. The input resistance

was monitored, and the recording was abandoned if it changed more than 15%. All signals were

recorded using an amplifier (MultiClamp700B; Axon Instruments Inc., Union City, CA) at a

holding potential of -60 mV, filtered at 1-2 kHz, digitized at 10 kHz, and stored into a computer

with pCLAMP 9.2 (Axon Instruments Inc.). DAMGO, naloxone, and GDP-β-S were obtained

from Sigma-Aldrich (St. Louis, MO). TTX and QX314 was obtained from Alomone Labs

(Jerusalem, Israel). Drugs were dissolved in Krebs solution and perfused into the tissue chamber

using syringe pumps.

Data analysis and statistics

Data are presented as means ± S.E.M. The amplitudes of evoked EPSCs and GIRK

currents were analyzed off-line with Clampfit 9.2 (Axon Instruments Inc.). The dose-response

effects of DAMGO on evoked EPSCs, mEPSCs, and GIRK currents were determined by either

two-way or repeated measures analysis of variance. The ED50 value of the DAMGO effect was

determined by nonlinear regression analysis of the concentration–response data using Prism

(GraphPad Software, San Diego, CA). Differences in the duration of DAMGO’s effect on GIRK

currents were analyzed using Student's t-test. Differences with P < 0.05 were considered to be


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statistically significant.


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Results

Effect of RTX on the thermal nociceptive threshold

Systemic injection of RTX caused a large increase in the paw-withdrawal threshold in

response to the noxious heat stimulus. In all of the RTX-treated rats, there was a large increase

in the paw withdrawal threshold in response to the heat stimulus as tested one week after RTX

injection (24.32 ± 2.34 vs. 9.66 ± 1.42 s pre-treatment, n = 18, P < 0.05). The impaired thermal

nociception persisted for at least 4 weeks after RTX injection. In contrast, systemic injection of

the vehicle did not significantly alter the paw withdrawal threshold in response to the noxious

heat (9.48 ± 1.41 vs. 9.51 ± 1.38 s pre-treatment, n = 16).

Effect of RTX on TRPV1 and IB4 labeling in the DRG and spinal cord

TRPV1 immunoreactivity and IB4-labeling were predominantly present in small- and

medium-sized DRG neurons in vehicle-treated rats. IB4-labeling was present in the majority of

TRPV1-immunoreactive DRG neurons (Fig. 1A). In RTX-treated rats, the TRPV1-

immunoreactive DRG neurons were largely eliminated (Fig. 1A). IB4-positive neurons were also

substantially reduced in the DRG sections from RTX-treated rats. The loss of TRPV1- and IB4-

positive DRG neurons by RTX treatment has been quantified in our previous study (Chen and

Pan, 2006b).

In vehicle-treated rats, TRPV1-immunoreactive- and IB4-positive terminals were present

in the superficial dorsal horn of the spinal cord (Fig. 1B). Dense TRPV1 immunoreactivity was

seen both at the dorsal root entry zone and in laminas I and II. TRPV1 immunoreactivity


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appeared largely co-localized on the same terminals labeled with IB4 in the laminas I and II of

rats treated with the vehicle. By contrast, the TRPV1-immunoreactive terminals were

completely abolished in the spinal dorsal horn of RTX-treated rats (Fig. 1B). Also, IB4-positive

terminals were markedly decreased in the superficial spinal dorsal horn of RTX-treated rats.

Notably, in the vehicle-treated rats, there was a subpopulation of TRPV1-immunoreactive dorsal

horn neurons. Similar to the results in our previous study (Chen and Pan, 2006b), these TRPV1-

immunoreactive neurons in the spinal dorsal horn were not affected in RTX-treated rats.

Effect of DAMGO on evoked glutamatergic EPSCs in the spinal cords of vehicle- and

RTX-treated rats

We first determined the effect of DAMGO on evoked glutamatergic EPSCs from primary

afferents to spinal lamina II neurons (evoked monosynaptic EPSCs) in vehicle- and RTX-treated

rats. The concentration-response effect was tested by bath application of 0.2-2 µM DAMGO to

the slice chamber in a cumulative fashion. Each DAMGO concentration was applied for 2 min.

The baseline amplitude of evoked monosynaptic EPSCs was significantly smaller in RTX-

treated than in vehicle-treated rats (Fig. 2). DAMGO significantly inhibited the amplitude of

evoked monosynaptic EPSCs in a concentration-dependent manner in both vehicle- and RTX-

treated rats (Fig. 2). The ED50 values for the effect of DAMGO on monosynaptic EPSCs were

0.38 ± 0.06 (n = 12 cells) and 0.34 ± 0.05 µM (n = 12 cells, P > 0.05, t-test) in vehicle- and

RTX-treated rats, respectively. The maximal inhibitory effect of DAMGO on monosynaptic

EPSCs was not significantly different between the vehicle-treated (62.2 ± 5.3%) and RTX-

treated (58.7 ± 4.7%) groups.


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In separate groups of lamina II neurons, DAMGO also concentration dependently

inhibited the amplitude of evoked polysynaptic EPSCs (to measure glutamate release from

primary afferents and interneurons) in vehicle-treated (83.9 ± 4.1%, n = 12 cells) and in RTX-

treated (93.0 ± 2.7%, n = 9 cells, P > 0.05) rats (Fig. 3). The ED50 value for the effect of

DAMGO on polysynaptic EPSCs was not significantly different between vehicle-treated and

RTX-treated rats (0.32 ± 0.05 vs. 0.29 ± 0.04 µM, P > 0.05) . The evoked monosynaptic and

polysynaptic EPSCs were completely blocked by the non-NMDA receptor antagonist CNQX (10

µM, data not shown). Also, the inhibitory effect of 1 µM DAMGO on evoked EPSCs in vehicle-

treated (n = 6 cells) and RTX-treated (n = 8 cells) rats was abolished by 1 µM naloxone, a

specific opioid receptor antagonist.

Because DAMGO’s inhibitory effect on synaptic glutamate release persisted after

washout in RTX-treated rats (Figs. 2 and 3), we next determined the duration of the effect of

DAMGO on evoked monosynaptic EPSCs in vehicle- and RTX-treated rats. In the vehicle-

treated group, bath perfusion of 1 µM DAMGO for 3 min decreased the amplitude of the evoked

monosynaptic EPSCs by 49.4 ± 5.7% (n = 13 cells). The amplitude of EPSCs gradually

returned to the baseline level within 7 min of DAMGO washout in vehicle-treated rats (Fig. 4, A

and B). In 6 of 13 lamina II neurons, there was an immediate rebound of evoked EPSCs after

washout of DAMGO. In RTX-treated rats, bath application of 1 µM DAMGO for 3 min

produced a similar inhibitory effect (52.2 ± 3.0%; n = 12 cells; P > 0.05) on the amplitude of

evoked monosynaptic EPSCs. Strikingly, in these rats, DAMGO’s inhibitory effect on the

amplitude of EPSCs persisted, with current values remaining below the baseline for at least 15

min after DAMGO washout (Fig. 4, A and B). In another 8 lamina II neurons (4 monosynaptic


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and 4 polysynaptic EPSCs) from RTX-treated rats, we found that when the spinal slices were

pretreated with 1 µM naloxone, 1 µM DAMGO failed to change significantly the amplitude of

evoked EPSCs.

Lack of reversing effect of naloxone on DAMGO-produced persistent inhibition on evoked

glutamate release in the spinal cords of RTX-treated rats

To determine whether the increased duration of the presynaptic effect of the µ-opioid

receptor agonist in RTX-treated rats is the result of increased binding affinity of the µ-opioid

receptors, we examined the effect of naloxone on the DAMGO-induced inhibition of evoked

monosynaptic EPSCs. In 8 additional lamina II neurons from RTX-treated rats, we initially

applied 1 µM DAMGO for 3 min and then bath applied 1 µM naloxone at 6 min after DAMGO

washout. Subsequent application of naloxone did not significantly reverse the long-lasting

effect of DAMGO on the amplitude of evoked EPSCs (Fig. 4B).

Effect of DAMGO on quantal glutamate release in the spinal cords of vehicle- and RTX-

treated rats

We further examined the presynaptic effect of DAMGO on quantal (action potential-

independent) glutamate release in the spinal cord of vehicle- and RTX-treated rats. The baseline

amplitude and frequency of mEPSCs were not significantly different between RTX-treated and

vehicle-treated rats. Each DAMGO concentration was applied for 2 min. Bath application of

0.2-2 µM DAMGO (each concentration was applied for 2 min) concentration-dependently

inhibited the frequency, but not the amplitude, of mEPSCs in both vehicle and RTX-treated


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groups (Fig. 5). The ED50 of the effect of DAMGO on the frequency of mEPSCs was 0.28 ±

0.04 µM (n = 8) and 0.33 ± 0.03 µM (n = 10, P > 0.05) in the vehicle-treated and RTX-treated

groups, respectively. The mEPSCs were completely blocked by 10 µM CNQX.

In separate groups of lamina II neurons, we also determined the duration of the inhibitory

effect of DAMGO on mEPSCs in vehicle- and RTX-treated rats. In the vehicle group, bath

perfusion of DAMGO (1 µM) for 3 min reduced the frequency of mEPSCs by 48.5 ± 2.7% (n =

12). The frequency of mEPSCs gradually returned to the basal level approximately 8 min after

DAMGO washout (Fig. 6). Bath perfusion of DAMGO (1 µM) for 3 min produced a similar

inhibitory effect (43.9 ± 4.2%; n = 9, P > 0.05) on the frequency of mEPSCs in RTX-treated

rats. However, DAMGO produced a sustained inhibition of the frequency of mEPSCs, and this

effect did not recover for at least 15 min after DAMGO washout in RTX-treated rats (Fig. 6).

Postsynaptic effect of DAMGO in the spinal cords of vehicle- and RTX-treated rats

We then conducted experiments to determine whether the postsynaptic effect of the µ-

opioid receptor agonist in the spinal cord of RTX-treated rats differed from that in control rats.

Because the µ-opioid receptor agonist can produce GIRK currents in many lamina II neurons

(Marker et al., 2006), we used the GIRK current as a measure for the postsynaptic effect of the

µ-opioid receptor agonist. Bath application of 0.2-2 µM DAMGO (each concentration was

applied for 1 min followed by washout to baseline) evoked an outward current in a

concentration-dependent manner in both vehicle- and RTX-treated groups (Fig. 7). In the

vehicle group, 40.6% (13 of 32 cells) of lamina II neurons exhibited an outward current after

DAMGO application. In RTX-treated rats, DAMGO application produced an outward current


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in 54.8% (23 of 42 cells) of lamina II neurons tested (P > 0.05, Fisher’s exact test). The

amplitude of the GIRK current produced by DAMGO was not significantly different between the

vehicle-treated and RTX-treated groups. DAMGO at 2 µM produced a maximal current

response in both vehicle-treated (29.8 ± 2.3 pA; n = 11 cells) and in RTX-treated (34.7 ± 2.7

pA; n = 17 cells) rats (P > 0.05). The DAMGO-elicited GIRK currents were completely blocked

in the presence of the opioid receptor antagonist naloxone (1 µM) or when 1 mM GDP-β-S was

included in the pipette internal solution for all the lamina II neurons examined (data not shown).

To further examine the duration of the effect of DAMGO on GIRK currents, we

examined the effect of bath application of 1 µM DAMGO for 3 min in separate groups of lamina

II neurons in vehicle-treated and RTX-treated rats. The duration of DAMGO’s effect on GIRK

currents was measured from the beginning of the GIRK current to the time at which the current

decay reached 20% of the maximal current. Upon washout of DAMGO, its effect on GIRK

currents rapidly disappeared within 6 min in both groups. The duration of DAMGO-produced

GIRK currents was not significantly different between the vehicle-treated (4.66 ± 0.46 min, n =

12 cells) and RTX-treated (5.49 ± 0.44 min, n = 16 cells) rats (Fig. 8).


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Discussion

The most salient finding of the present study is that activation of µ-opioid receptors

produced long-lasting inhibition of synaptic glutamate release from primary afferent terminals

when TRPV1-expressing afferent terminals were removed. We found that the concentration-

response and maximal inhibition of evoked monosynaptic and polysynaptic EPSCs and mEPSCs

by the µ-opioid receptor agonist DAMGO were not significantly different between vehicle- and

RTX-treated rats. However, briefly applying DAMGO to the spinal cord slices caused a striking

prolongation of its inhibitory effect on monosynaptic evoked EPSCs and mEPSCs in RTX-

treated rats. Also, naloxone did not readily reverse the prolonged presynaptic effect of DAMGO

in RTX-treated rats, which is consistent to our previous finding that the binding affinity of the

µ-opioid receptors is not altered by RTX treatment (Chen and Pan, 2006b). We observed that

the concentration-response of GIRK currents evoked by DAMGO and the duration of the effect

were similar in vehicle- and RTX-treated rats. Our study demonstrates that stimulation of the µ-

opioid receptors produces a long-lasting inhibition of glutamate release from non-TRPV1-

expressing primary afferent terminals. Our findings also suggest that the desensitization process

of µ-opioid receptors located on TRPV1- and non-TRPV1-expressing nociceptive afferent

terminals may be distinctly different.

Systemic administration of RTX induces a substantial reduction in presynaptic µ-opioid

receptors in the spinal cord because TRPV1-expressing afferent neurons are lost (Chen and Pan,

2006b). However, the analgesic effect of the µ-opioid receptor agonists, morphine and

DAMGO, is potentiated in RTX-treated rats (Chen and Pan, 2006b). RTX treatment also


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attenuates the development of morphine analgesic tolerance (Chen et al., 2007). The cellular

and molecular mechanisms underlying the altered opioid action in RTX-treated rats are little

known. The µ-opioid receptor agonists inhibit glutamatergic synaptic transmission in the spinal

dorsal horn (Kohno et al., 1999). The presynaptic effect of µ-opioid receptor agonists is

mediated by their inhibitory effect on voltage-gated Ca2+ channels (Rusin and Moises, 1995; Wu

et al., 2004) and the direct inhibitory effect on the transmitter release machinery, by binding to

proteins of the SNARE complex (Blackmer et al., 2005; Gerachshenko et al., 2005).

Postsynaptically, the µ-opioid receptor agonists inhibit the excitability of spinal dorsal horn

neurons by opening GIRK channels (Schneider et al., 1998; Marker et al., 2006). These

combined presynaptic and postsynaptic effects of µ-opioid receptor agonists contribute to their

analgesic action in the spinal cord. We therefore examined the presynaptic and postsynaptic

effects of the µ-opioid receptor agonist in the spinal cord to determine the cellular mechanisms

underlying potentiated opioid analgesia after removing TRPV1-expressing afferent neurons. We

observed that the baseline amplitude of EPSCs was significantly smaller in RTX-treated than in

vehicle-treated rats. Our immunocytochemical studies documented the reduction in IB4-positive

afferent terminals and the depletion of TRPV1 immunoreactivity in the DRG and superficial

dorsal horn of RTX-treated rats. Also, the histological study has shown that RTX treatment

causes a large reduction in unmyelinated fibers in the sciatic nerve (Pan et al., 2003). Thus, the

reduced glutamatergic input evoked from primary afferents is likely due to the removal of

TRPV1-expressing afferent terminals in the spinal dorsal horn by RTX treatment. Despite the

fact that the number of µ-opioid receptors in the spinal cord is significantly reduced in RTX-

treated rats, as shown by our previous study (Chen and Pan, 2006b), we found that DAMGO


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inhibited evoked monosynaptic and polysynaptic glutamatergic EPSCs and mEPSCs in a

concentration-dependent manner, and that these effects were not significantly different between

vehicle- and RTX-treated rats. These data suggest that stimulation of the remaining µ-opioid

receptors on non-TRPV1-expressing primary afferents can potently inhibit neurotransmitter

release to spinal dorsal horn neurons. Because DAMGO similarly inhibited EPSCs evoked from

the primary afferents in vehicle-treated and RTX-treated rats, it is unlikely that the increased

opioid analgesia in RTX-treated rats was due to the difference in the µ-opioid receptor density

between TRPV1- and non-TRPV1-expressing primary afferents.

To our knowledge, this is the first study demonstrating that activation of µ-opioid

receptors can cause sustained inhibition of glutamate release from non-TRPV1-expressing

primary afferent terminals in the spinal cord. We found that in vehicle-treated rats, brief

exposure of the spinal cord slices to DAMGO inhibited evoked monosynaptic EPSCs, and this

effect disappeared rapidly after drug washout. In contrast, the DAMGO-induced inhibition of

monosynaptic EPSCs in RTX-treated rats became long-lasting, persisting for at least 15 min

after DAMGO washout. When the TRPV1-expressing primary afferents were intact, we found

that there was an immediate rebound of evoked EPSCs in some lamina II neurons after washout

of DAMGO. Thus, the sustained inhibitory effect of DAMGO on synaptic glutamate release

from non-TRPV1-expressing afferents might be masked by the presence of TRPV1-expressing

afferent terminals. The precise cellular mechanisms underlying this prolonged presynaptic

action in RTX-treated rats remain to be determined. Because subsequent treatment with

naloxone failed to reverse the long-lasting inhibitory effect of DAMGO on evoked EPSCs in

RTX-treated rats, it is less likely that the prolonged presynaptic effect of DAMGO is due to


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increased binding affinity of surface µ-opioid receptors in the spinal cord by RTX treatment.

We also found that DAMGO caused a persistent inhibition of the frequency of mEPSCs, a

measure of quantal vesicle release independent of voltage-activated Ca2+ channels, in spinal

dorsal horn neurons of RTX-treated rats. The effect of the µ-opioid receptor agonist on mEPSCs

is possibly due to direct inhibition of the vesicle release machinery, such as SNAP25 (Capogna

et al., 1993; Gerachshenko et al., 2005). Our findings suggest that the prolonged inhibitory

effect of the µ-opioid receptor agonist at non-TRPV1-expressing afferent terminals is not the

results of increased coupling between the µ-opioid receptor and voltage-activated Ca2+ channels.

It is known that the µ-opioid receptors are desensitized/inactivated by G protein-coupled

receptor kinases (Celver et al., 2004), β-arrestins (Bohn et al., 1999; Bohn et al., 2000), and

protein kinase C (PKC) (Narita et al., 2001). In β-arrestin2-knockout mice, presynaptic

inhibition of GABA release by the µ-opioid receptor agonist is enhanced, whereas the

postsynaptic GIRK currents elicited by the µ-opioid receptor agonist are not affected in the locus

coeruleus and periaqueductal gray (Bradaia et al., 2005). Thus, if β-arrestins are inadequately

expressed in TRPV1-negative primary afferents, it could explain why the presynaptic inhibitory

effect of the µ-opioid receptor agonist in the spinal cord is sustained in RTX-treated rats.

Furthermore, the analgesic effect produced by µ-opioid receptor agonists is potentiated in PKC-

knockout mice (Narita et al., 2001). Because PKC is mainly expressed in IB4-positive (mostly

TRPV1-expressing) DRG neurons (Molliver et al., 1995), the differential expression of PKC in

TRPV1- and non-TRPV1-sensory neurons may profoundly influence the rate of µ-opioid

receptor desensitization/inactivation. We have shown that morphine analgesic tolerance is

attenuated in RTX-treated rats (Chen et al., 2007). If RTX treatment predominantly removes


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TRPV1-containing afferent terminals in which the µ-opioid receptors are co-expressed with a

high level of PKCγ (Chen et al., 2007), it could prolong the presynaptic effect of the µ-opioid

receptor agonist. Further studies are warranted to determine the differential expression of the

signaling molecules involved in µ-opioid receptor desensitization in TRPV1- and non-TRPV1-

expressing sensory neurons.

Activation of GIRK channels by the µ-opioid receptor agonist can reduce neuronal

excitability and inhibit nociceptive transmission in the spinal cord (Schneider et al., 1998;

Marker et al., 2006). This postsynaptic effect contributes to the analgesic action of the µ-opioid

receptor agonists (Ikeda et al., 2000; Marker et al., 2005). Because removal of TRPV1-

expressing primary afferents may cause an upregulation of postsynaptic µ-opioid receptors, we

further examined the postsynaptic effect of the µ-opioid receptor agonist in the spinal cord of

RTX-treated rats. We observed that the concentration-response and the duration of effects of

DAMGO on GIRK currents were not significantly different between vehicle-treated and RTX-

treated rats. Thus, it is less likely that the potentiated analgesic effect of the µ-opioid receptor

agonist in RTX-treated rats is due to changes in the postsynaptic µ-opioid receptors.

In summary, the present study demonstrates that deleting TRPV1-expressing sensory

neurons preferentially increases the duration of the presynaptic inhibitory effect of the µ-opioid

receptor agonist in the spinal cord. This persistent presynaptic inhibition by the µ-opioid

receptor agonist could explain the prolonged duration of opioid analgesia found when TRPV1-

expressing primary afferents are absent (Chen and Pan, 2006b). Our findings provide a cellular

basis for the possible differential signaling of µ-opioid receptors in different populations of

nociceptive primary afferent terminals. This information is also important for our understanding


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of how µ-opioid receptor agonists produce a more prolonged analgesic effect on mechanical

nociception than thermal (TRPV1-mediated) nociception, which may ultimately improve the

clinical use of opioid therapy for different pain states.


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Footnotes

This study was supported by grants GM64830 and NS45602 from the National Institutes

of Health.


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Legends for Figures

Fig. 1. Confocal images showing the effect of RTX on TRPV1- and IB4-positive dorsal root

ganglion (DRG) neurons and afferent terminals in the spinal cord. A: representative confocal

images showing TRPV1 (green)-immunoreactive and IB4-positive (red) DRG neurons from one

vehicle-treated and one RTX-treated rat. B: confocal images showing TRPV1 (green)-

immunoreactive and IB4-positive (red) afferent terminals in the spinal dorsal horn of one

vehicle- and one RTX-treated rat. Co-localization of TRPV1 immunoreactivity and IB4 labeling

is indicated in yellow when two images are digitally merged. All images are single confocal

optical sections.

Fig. 2. Effect of DAMGO on evoked monosynaptic EPSCs of lamina II neurons in vehicle-

treated and RTX-treated rats. A, original current traces showing the effect of different

concentrations (0.2-2 µM) of DAMGO on monosynaptic EPSCs of lamina II neurons in one

vehicle- and one RTX-treated rat. B, concentration-response curve of the effect of 0.2-2 µM

DAMGO on the peak amplitude (left) and percentage of inhibition (right) of monosynaptic

EPSCs of lamina II neurons in vehicle-treated (n = 12 cells) and RTX-treated (n = 12 cells) rats.

*, P < 0.05 compared with the control value. #, P < 0.05 compared with the corresponding

value in the vehicle group.

Fig. 3. Effect of DAMGO on evoked polysynaptic EPSCs of lamina II neurons in vehicle-

treated and RTX-treated rats. A, representative traces showing the effect of 0.2-2 µM DAMGO


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on the peak amplitude (left) and percentage of inhibition (right) of polysynaptic EPSCs in one

vehicle- and one RTX-treated rat. B, concentration-response curve of the effect of 0.2-2 µM

DAMGO on the amplitude of polysynaptic EPSCs of lamina II neurons in vehicle-treated (n =

12 cells) and RTX-treated (n = 9 cells) rats. *, P < 0.05 compared with the control value. #, P <

0.05 compared with the corresponding value in the vehicle group.

Fig. 4. Durations of the inhibitory effect of DAMGO (1 µM for 3 min) on evoked monosynaptic

EPSCs of lamina II neurons in vehicle-treated and RTX-treated rats. A, original traces showing

the monosynaptic EPSCs during the control period, during application of 1 µM DAMGO, and at

different time points after DAMGO washout in one vehicle- and one RTX-treated rat. B, time

course of the persistent inhibitory effect of 1 µM DAMGO after washout on the peak amplitude

of monosynaptic EPSCs of lamina II neurons in vehicle-treated (n = 13 cells) and RTX-treated

(n = 12 cells) rats. Note that subsequent application of 1 µM naloxone for 3 min did not reverse

the persistent inhibitory effect of 1 µM DAMGO in another 8 lamina II neurons from RTX-

treated rats. *, P < 0.05 compared with the respective control value. #, P < 0.05 compared with

corresponding value in the vehicle group.

Fig. 5. Effect of DAMGO on mEPSCs in lamina II neurons in vehicle-treated and RTX-treated

rats. A, original traces showing the effect of different concentrations of DAMGO on mEPSCs in

a lamina II neuron from a vehicle-treated and an RTX-treated rat. B, concentration-response

curves showing the inhibitory effect of DAMGO on the frequency of mEPSCs in lamina II

neurons from the vehicle treated and RTX-treated rats. *, P < 0.05 compared with the


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respective control. Cont, control.

Fig. 6. Duration of the inhibitory effect of DAMGO on mEPSCs in lamina II neurons from

vehicle-treated and RTX-treated rats. A, original traces showing the time course and duration of

the DAMGO (1 µM for 3 min)-induced changes in the mEPSC frequency in a lamina II neuron

from a vehicle-treated and an RTX-treated rat. B, summary data showing the duration of the

inhibitory effect of DAMGO (1 µM for 3 min) on the frequency of mEPSCs in lamina II neurons

from the vehicle-treated and RTX-treated rats. *, P < 0.05 compared with the respective control.

Cont, control.

Fig. 7. Effect of DAMGO on postsynaptic GIRK currents of lamina II neurons in vehicle-treated

and RTX-treated rats. A, original current traces showing the effect of different concentrations

(0.2-2 µM) of DAMGO on GIRK currents of lamina II neurons in one vehicle-treated and one

RTX-treated rat. B, concentration-response curve of the inhibitory effect of 0.2-2 µM DAMGO

on the peak amplitude of GIRK currents of lamina II neurons in vehicle-treated (n = 11 cells)

and RTX-treated (n = 17 cells) rats. *, P < 0.05 compared with the respective control value.

Fig. 8. Duration of the effect of DAMGO (1 µM for 3 min) on GIRK currents of lamina II

neurons in vehicle-treated and RTX-treated rats. A, original recordings showing the GIRK

current of lamina II neurons produced by application of 1 µM DAMGO in one vehicle-treated

and one RTX-treated rat. B, comparison of the duration of the effect of 1 µM DAMGO on

GIRK currents of lamina II neurons in vehicle-treated (n = 12 cells) and RTX-treated (n = 16


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cells) rats.


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