gregory scherrer, noritaka imamachi, yu-qing cao, … · ... yu-qing cao, candice contet,...

Cell, Volume 137

Supplemental Data

Dissociation of the Opioid Receptor

Mechanisms that Control

Mechanical and Heat Pain Gregory Scherrer, Noritaka Imamachi, Yu-Qing Cao, Candice Contet, Françoise Mennicken, Dajan O’Donnell, Brigitte L. Kieffer, and Allan I. Basbaum

Supplementary Table 1.

Neurochemistry of the DOReGFP DRG neurons

Cross sectional area % of DOReGFP cells that coexpress: 100 – 400 μm2 400 – 1400 μm2 Substance P 4.5 ± 0.9 - CGRP 25.7 ± 6.6 42.6 ± 5.5 TRPV1 12.1 ± 3.6 - IB4 91.8 ± 1.6 - P2X3 91.0 ± 5.1 - NF200 - 92.8 ± 1.9 TRPV2 - 64.6 ± 4.7

Cross sectional area % of cells that coexpress DOReGFP 100 – 400 μm2 400 – 1400 μm2 Substance P 1.6 ± 0.4 - CGRP 8.8 ± 2.7 55.5 ± 6.1 TRPV1 1.4 ± 0.9 - IB4 15.7 ± 1.3 - P2X3 12.7 ± 5.7 - NF200 - 26.0 ± 2.1 TRPV2 - 37.6 ± 2.9

Legend to supplementary figures.

Supplementary figure 1. Colocalization of DOReGFP and P2X3 in DRG cell bodies.

Small diameter cells that express DOReGFP coexpress the purinergic receptor P2X3

(arrows in the merged image), and thus belong to the nonpeptidergic subset of

unmyelinated primary afferent fibers. Scale bar equals 20 µm.

Supplementary figure 2. The binding pattern obtained with DOR selective ligands

matches the distribution of DOReGFP in the spinal cord.

The binding pattern using [125I]-deltorphin II or [125I]-DPDPE is comparable to that

revealed in the DOReGFP mice, (Figure 2), with dense labeling in the superficial dorsal

horn, as well as a relatively uniform lighter staining throughout the grey matter of the

spinal cord.

(A) Autoradiogram using [125I]-deltorphin II.

(B) Autoradiogram using [125I]-DPDPE.

Supplementary figure 3. As for the bladder, the kidney is densely innervated by

substance P-immunoreactive fibers (red, middle panel) but is almost completely devoid

of DOReGFP positive terminals (green, left panel). Quantitative analysis in the kidney

revealed a mean of 39.7 ± 2.6 substance P positive fibers/mm versus 1.5 ± 1.0

DOReGFP positive fibers/mm, p < 0.001 (Student’s t-test).

Supplementary figure 4. Immunostaining pattern obtained with the widely used 3-17

N-terminal anti-DOR antibody (Chemicon/Millpore AB5503).

(A) High power image showing a granular staining pattern in small diameter DRG

neurons, suggesting that the epitope recognized by the anti-DOR antibody is localized

in large dense core vesicles. Scale bar, 20 µm.

(B, C) Double immunostaining illustrates that immunoreactivity arising from the anti-

DOR antibody colocalizes with substance P, both in cell bodies of the DRG (B) and in

spinal cord terminals (C), in agreement with the literature (Bao et al., 2003; Dado et al.,

1993)

Supplementary figure 5 The 3-17 N-terminal anti-DOR antibody recognizes a

molecule other than the DOR. (A) The pattern of immunostaining with the traditional

anti-DOR antibody does not differ in two strains of dor knockout mice, compared to wild

type mice (Filliol et al., 2000; Zhu et al., 1999).

(B) Quantification of results in A; mean ± SEM, n=8/group (Student’s t-test).

Supplementary figure 6. Persistence of the staining of three additional anti-DOR

antibodies in spinal cord sections from dor null mutant mice. Note that these antibodies

generate qualitatively distinct immunoreactivity patterns indicating that they recognize

different molecules other than DOR.

(A) 3-17 N-terminus directed antibody (AB1560), which differs from the original 3-17

anti-DOR antibody (AB5503). Middle panel: superficial dorsal horn of the spinal cord,

right panel: ventral horn.

(B) Anti-DOR antibody raised against the first extracellular loop (amino acids 111-120,

OR-500). Right panel: superficial dorsal horn of the spinal cord.

(C) C-terminus directed anti-DOR antibody (amino acids 358-372, RA10101). Right

panel: superficial dorsal horn of the spinal cord.

All scale bars equal 50 µm.

Supplementary figure 7. DOR transport to the plasma membrane of DRG neurons is

independent of substance P.

(A) As for the spinal cord (Supplementary figure 3), immunostaining with the anti-DOR

antibody (AB5503) is lost in the DRG taken from ppt-A null mutant mice.

(B) RT-PCR analysis demonstrates that the level of dor transcript is unchanged in

trigeminal ganglion taken from ppt-A null mutant mice compared to wild-type mice.

(C) High power image of a rare DRG neuron coexpressing DOReGFP and substance P

(arrow, left panel) illustrates that DOReGFP predominates at the cell surface (arrow,

right panel) and does not colocalize with substance P, which is concentrated in LDCVs.

(D) Substance P is not required for the transport or the stabilization of DOReGFP at the

plasma membrane. Thus, DOReGFP is present at the plasma membrane of DRG

neurons even in the absence of substance P (in ppt-A knockout mice).


Supplementary figure 8. The anti-MOR antibody recognizes the MOR in the DRG and

mouse spinal cord.

(A, B) Immunoreactivity obtained with the anti-MOR antibody is lost in dorsal root

ganglia (A) and spinal cord (B) sections taken from mice with a deletion of the mor

gene. (Left: wild type; right; null mutant mice).

(C) Quantification of results in B; mean ± SEM, n=8/group; *** p < 0.001.

Supplementary figure 9. Segregated distribution of DOR and MOR in the spinal cord.

(A) Low power images of a spinal cord section from DOReGFP mice immunostained

with anti-GFP (left, green) and anti-MOR (middle, red) antibodies showing the

complementarity of the two labeling patterns. MOR staining is restricted to lamina I and

outer II (IIo), whereas DOReGFP is present throughout the grey matter, predominating

in laminae I and inner II (IIi) of the superficial dorsal horn.

(B) Although both the MOR and DOReGFP are located in lamina I, high power analysis

reveals only limited colocalization of the two receptors.


Supplemental References

Bao, L., Jin, S.X., Zhang, C., Wang, L.H., Xu, Z.Z., Zhang, F.X., Wang, L.C., Ning, F.S.,

Cai, H.J., Guan, J.S., et al. (2003). Activation of delta opioid receptors induces

receptor insertion and neuropeptide secretion. Neuron 37, 121-133.

Dado, R.J., Law, P.Y., Loh, H.H., and Elde, R. (1993). Immunofluorescent identification

of a delta (delta)-opioid receptor on primary afferent nerve terminals. Neuroreport

5, 341-344.

Filliol, D., Ghozland, S., Chluba, J., Martin, M., Matthes, H.W., Simonin, F., Befort, K.,

Gaveriaux-Ruff, C., Dierich, A., LeMeur, M., et al. (2000). Mice deficient for delta-

and mu-opioid receptors exhibit opposing alterations of emotional responses. Nat

Genet 25, 195-200.

Zhu, Y., King, M.A., Schuller, A.G., Nitsche, J.F., Reidl, M., Elde, R.P., Unterwald, E.,

Pasternak, G.W., and Pintar, J.E. (1999). Retention of supraspinal delta-like

analgesia and loss of morphine tolerance in delta opioid receptor knockout mice.

Neuron 24, 243-252.

Supplemental Results DOReGFP and SP distribution in visceral afferents. The segregated expression of DOR and SP is not restricted to somatic afferent

nociceptors. In fact, it is particularly apparent for afferents that innervate viscera. First,

although numerous cell bodies in the nodose ganglion, the axons of which innervate

visceral organs, express SP, almost none are DOReGFP-positive (Figure 2G). Second,

the central target of visceral afferents in the brainstem, namely the nucleus of the

solitary tract (NTS), receives a large input from SP-containing unmyelinated afferents

axons, but is devoid of DOReGFP innervation (Figure 2H). Consistent with this

somatic/visceral dissociation, analysis of the peripheral afferents innervating the bladder

revealed a dense network of SP fibers, but rarely did we find DOReGFP terminals

(Figure 2I). Together these results not only indicate that the DOR is not expressed in

SP-containing nociceptors, but also that the DOR is largely excluded from the

innervation of major visceral organs.

Specificity of anti-DOR antisera

Immunostaining of the DRG with the 3-17 amino terminal-directed anti-DOR antibody,

which is the most widely used, revealed a granular cytoplasmic staining consistent with

an LDCV localization (Figure S3A). Furthermore, DOR and SP immunoreactivity

colocalize in DRG neurons (Figure S3B) and in the spinal cord (Figure S3C), in

agreement with numerous reports in the literature (Bao et al., 2003; Dado et al., 1993;

Guan et al., 2005; Zhang et al., 1998).

As the patterns of DOR expression revealed with antisera or in the DOReGFP mice are

completely different, we tested for the specificity of the anti-DOR antibody by

immunostaining tissues from two different strains of DOR knockout mice, one with a

disruption of exon 1 (Filliol et al., 2000), another with a disruption of exon 2 (Zhu et al.,

1999). In both mutant mice, dor mRNA and CNS binding of DOR ligands are lost.

However, supplementary figures 4A and 4B show that neither the pattern nor the

intensity of dorsal horn immunostaining produced with the anti-DOR antibody differed in

the knockout mice, compared to their wild-type littermates. We conclude that this anti-

DOR antibody does not recognize the DOR in immunohistochemical preparations, but

rather must cross-react with an as yet unidentified molecule.

Development of tolerance to the analgesic effect of morphine in dor and ppt-A knockout

mice.

We reexamined the impact of the deletion of the ppt-A or the dor gene on morphine

tolerance (Figure G-I). We indeed found that inactivation of the ppt-A gene greatly

reduced analgesic tolerance to morphine, both in mice on the wild-type (Figure 6G) and

doregfp genetic backgrounds (Figure 6H). In contrast, analgesic tolerance to morphine

developed normally in mice with a deletion of the dor gene (Figure 6I). Together, our

results indicate that the decreased analgesic tolerance to morphine in ppt-A knockout

mice is not attributable to alterations in DOR expression or transport, but rather to the

loss of SP itself. It follows that regulation of the MOR in pain processing is not a primary

function of the DOR. The two receptors are differentially distributed in primary pain

fibers and selectively regulate different pain modalities. We propose that an interaction

of the two receptors is not required for the development of analgesic tolerance with

chronic morphine.


Bao, L., Jin, S.X., Zhang, C., Wang, L.H., Xu, Z.Z., Zhang, F.X., Wang, L.C., Ning, F.S.,

Cai, H.J., Guan, J.S., et al. (2003). Activation of delta opioid receptors induces

receptor insertion and neuropeptide secretion. Neuron 37, 121-133.

Dado, R.J., Law, P.Y., Loh, H.H., and Elde, R. (1993). Immunofluorescent identification

of a delta (delta)-opioid receptor on primary afferent nerve terminals. Neuroreport

5, 341-344.




Genet 25, 195-200.

Guan, J.S., Xu, Z.Z., Gao, H., He, S.Q., Ma, G.Q., Sun, T., Wang, L.H., Zhang, Z.N.,

Lena, I., Kitchen, I., et al. (2005). Interaction with vesicle luminal protachykinin

regulates surface expression of delta-opioid receptors and opioid analgesia.

Cell 122, 619-631.

Zhang, X., Bao, L., Arvidsson, U., Elde, R., and Hokfelt, T. (1998). Localization and

regulation of the delta-opioid receptor in dorsal root ganglia and spinal cord of the

rat and monkey: evidence for association with the membrane of large dense-core

vesicles. Neuroscience 82, 1225-1242.




Neuron 24, 243-252.

Supplemental Experimental Procedures Immunohistochemistry

Mice were deeply anesthetized with 100 mg/kg sodium pentobarbital and perfused

transcardially with 0.1 M phosphate buffered saline (PBS) followed by 10%

formalin in PB. Tissues were dissected, post-fixed in the same fixative for 4 hours,

and cryoprotected overnight in 30% sucrose in PBS. Brains and spinal cords were

sectioned on a cryostat at 40 µm and processed as free-floating sections. DRGs,

skin and bladder were cut at 25 µm and sections were processed on slides. Tissue

was incubated for at least 1 hour in 0.1 M PBS with 0.3% Triton X-100 (Sigma)

plus 5% normal goat serum (Antibodies Inc., Davis, CA, USA). Primary and

secondary antibodies were diluted in 0.1 M PBS with 0.3% Triton X-100 plus 1%

normal goat serum. Tissue was incubated overnight at RT in primary antibody,

washed and then incubated for a further 2 hours in secondary antibody. Images

were acquired with a Zeiss LSM510 Meta confocal microscope. We used the Zeiss

LSM image browser to measure the density of substance P+ and DOReGFP+

fibers in the bladder and kidney. ImageJ (NIH) was used for the quantification of

fluorescence intensities in the superficial dorsal horn of the spinal cord and for the

quantification of DOR internalization as described previously (Scherrer et al.).

We used the following primary antibodies: CGRP: Peninsula (1:1000).

DOR: Millipore (Chemicon): rabbit AB5503 (1:2000); Millipore (Chemicon): rabbit

AB1560 (1:1000); Neuromics: rabbit RA10101 (1:1000); Gramsch Laboratories:

OR-500 (1:1000).

GFP: Molecular Probes or Abcam (1:1000).

IB4: Sigma (1:500).

MOR: a gift from CJ. Evans (1:300).

NF200: Sigma (1:1000).

P2X3: Chemicon (1:7000)

PKCγ: Strategic Biosolutions (1:10000).

Substance P: a gift from J.E. Maggio (1:10,000).

TRPV1: a gift from D. Julius (1:1000).

TRPV2: a gift from D. Julius (1:2000).

Autoradiography

Serial coronal 16 µm-thick sections of lumbar spinal cord were cut on a cryostat

(Microm HM 500M, Germany) and mounted onto Probe-On Plus slides (Fisher

Scientific, Montreal, Quebec), and stored at -80°C until use. Serial sections were

processed for receptor autoradiography using [125I]-deltorphin II or [125I]-DPDPE

(Plobeck et al., 2000; iodination was performed using the chloramine T method;

specific activity was considered to be 2000 Ci/mmol, the theoretical value).

Sections were preincubated for 30 min at room temperature in 50 mM Tris-HCl,

120 mM NaCl, and 1 mM MgCl2 buffer (pH 7.4) and then incubated for 60

minutes at room temperature in the same buffer containing 0.5% bovine serum

albumin (BSA), 0.1 mM Bestatin, 0.1 mM PMSF, and 40 pM [125I]-deltorphin II or

[125I]-DPDPE. Non-specific binding was determined in the presence of 100 nM

diprenorphine for both radioligands. Sections were washed 3 X 3 min in ice-cold

Tris-HCl buffer, air dried, and exposed to Kodak BioMax MS film for 2 weeks.

Film autoradiograms were digitized using the MCID image analysis system

(Imaging Research Inc., St-Catharines, Ontario) equipped with a high resolution

Xillix Microimager digital camera (Xillix Technologies Corp., Richmond, British

Columbia). For each mouse, 5 to 12 sections were used for quantifying labeling

intensity in the superficial dorsal horn (laminae I-II). The intensity of labeling was

expressed in optical density units and the data obtained from the left and right

sides were averaged for each section. Statistical analyses were then performed

using one-way ANOVA followed by Fisher Post-hoc test to compare directly

signal intensities in ppt-A mutant mice and wild-type littermates for each

radioligand.

RT-PCR

Adult wild-type and ppt-A knockout mice were euthanized by cervical dislocation

and trigeminal ganglia were quickly dissected out, pooled and stored on dry ice.

Total RNA was extracted with Trizol (Invitrogen). Five μg of RNA from each

group was used for the first-strand cDNA synthesis with SuperScript reverse

transcriptase (Invitrogen); 1/20 of the first-strand cDNA was used as template to

amplify a 600 bp fragment from mouse DOR cDNA using the Expand High

Fidelity DNA polymerase (Roche). Twenty cycles of PCR were performed to

ensure linear amplification. To avoid amplification from genomic DNA, the

primers were designed to expand the >7kb intron between exons 1 and 2

(forward primer: 5’-CTCGTCAACCTCTCGGACGCCTTT-3’ and reverse primer:

5’-GAGGAACACGCAGATCTTGGTCACA-3’).

Behavior

Animals

Adult male C57Bl6 mice (20-30 g) were purchased from Charles River (Hollister,

CA, USA), housed 2-5 per cage and maintained on a 12 hour light/dark cycle in a

temperature-controlled environment with ad lib access to food (Purina LabDiet,

Richmond, IL, USA) and water. Behavioral testing was performed after at least one

week of acclimation to the housing facility. The generation of DOReGFP mice as

well as dor and ppt-A mutant mice have been described earlier (Cao et al., 1998;

Filliol et al., 2000; Scherrer et al., 2006; Zhu et al., 1999). All animal experiments

were approved by the Institutional Animal Care and Use Committee of the

University of California at San Francisco and were conducted in accordance with

the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Intrathecal injections of capsaicin.

Mice were anesthetized with 1.5% isoflurane prior to, during, and for 30 min after

the injection. Intrathecal (i.t.) injection of capsaicin (10 µg; SIGMA, St. Louis, MO,

USA) or vehicle (10% ethanol, 10% Tween 80, saline) in a volume of 5.0 µl was

achieved by direct injection at the level of the pelvic girdle with a luer-tipped

Hamilton syringe to which a 30 gauge needle was attached. After the injection, we

placed the mice under a heat lamp to prevent hypothermia. Absence of a response

on the hot plate and loss of TRPV1 immunoreactivity in the superficial dorsal horn

seven days after the injection confirmed accurate placement of the intrathecal

injection and appropriate destruction of TRPV1-expressing terminals.

Intrathecal injections of opioid compounds

Animals were placed in habituation boxes one hour prior to the administration of

opioid compounds or their vehicle solutions. We injected 5.0 µl intrathecally in

unanesthetized mice, as above. Flick of the tail confirmed appropriate placement of

the needle. SNC80 (Tocris bioscience, Ellisville, MI, USA) was dissolved in sterile

acidic (0.2% HCl) saline solution. DAMGO (Sigma, St. Louis, MO, USA) and

naltrindole (NTI, Sigma, St. Louis, MO, USA) were dissolved in sterile saline

solution. Behavioral testing was performed 15 minutes after drug administration by

an individual blind to the treatments.

Tail immersion test

The mouse was gently maintained in a towel and the tip of the tail immersed in

50ºC water. We monitored the latency for tail withdrawal. Cutoff latency to avoid

tissue damage was 15 sec.

Hindpaw radiant heat (Hargreaves’) test

Mice were placed in plastic chambers on a glass surface heated to 25ºC through

which a radiant heat source (Dept of Anesthesiology, UC San Diego, La Jolla, CA)

could be focused on the hindpaw. Latency to withdraw the paw was measured as

the average of at least 3 trials per animal taken ≥ 5 min apart. Cutoff latency to

avoid tissue damage was 20 sec.

von Frey test of mechanical threshold

Mice were placed in plastic chambers on a wire mesh grid and stimulated with von

Frey filaments (North Coast Medical Inc., Morgan Hill, CA, USA) according to the

up-down method (Chaplan et al., 1994), starting with 0.1 g and ending with 2.0 g

filament as cutoff value.

Complete Freund's Adjuvant (CFA)-induced heat and mechanical hyperalgesia

We made an intraplantar injection of 10 µl of a 1:1 saline/CFA (Sigma, St. Louis,

MO, USA) emulsion with a 30 gauge needle. Thermal and mechanical thresholds

were measured using the hindpaw radiant heat and von Frey tests, as described

above, 4 days after the injection.

Spared nerve injury model of neuropathic pain

We performed the spared nerve injury surgery as described (Shields et al., 2003).

Briefly, mice were maintained at 2% isoflurane anesthesia while an incision was

made through the skin and thigh muscle at the level of the trifurcation of the sciatic

nerve. The common sural and peroneal nerve were ligated with 8-0 silk (Ethicon,

Piscataway, NJ, USA) and transected, leaving the tibial nerve intact. Mechanical

threshold was determined as described above, 7 days after the surgery.

Analgesic tolerance to morphine

Mice were injected twice daily (9:00 and 19:00) with morphine (20 mg/kg; i.p.).

Morphine-induced analgesia was monitored daily using the tail immersion test,

performed 45 min after the morning injection. The dose of morphine, the route of

administration, and the testing time were chosen following pilot experiments.


Cao, Y.Q., Mantyh, P.W., Carlson, E.J., Gillespie, A.M., Epstein, C.J., and Basbaum,

A.I. (1998). Primary afferent tachykinins are required to experience moderate to

intense pain. Nature 392, 390-394.

Chaplan, S.R., Bach, F.W., Pogrel, J.W., Chung, J.M., and Yaksh, T.L. (1994).

Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 53,

55-63.




Genet 25, 195-200.

Scherrer, G., Tryoen-Toth, P., Filliol, D., Matifas, A., Laustriat, D., Cao, Y.Q., Basbaum,

A.I., Dierich, A., Vonesh, J.L., Gaveriaux-Ruff, C., et al. (2006). Knockin mice

expressing fluorescent delta-opioid receptors uncover G protein-coupled receptor

dynamics in vivo. Proc Natl Acad Sci (PNAS) 103, 9691-9696.

Shields, S.D., Eckert, W.A., 3rd, and Basbaum, A.I. (2003). Spared nerve injury model

of neuropathic pain in the mouse: a behavioral and anatomic analysis. J Pain 4,

465-470.




Neuron 24, 243-252.

gregory scherrer, noritaka imamachi, yu-qing cao, … · ... yu-qing cao, candice contet,...

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