Equal proportions of small and large DRG neurons express opioid receptor mRNAs

Equal Proportions of Small and LargeDRG Neurons Express Opioid

Receptor mRNAs

HONG WANG AND MARTIN W. WESSENDORF*

Department of Neuroscience, University of Minnesota, Minneapolis, Minnesota 55455

ABSTRACTPrevious studies have reported that the mRNAs encoding the cloned m-opioid receptor

(MOR1) and the cloned d-opioid receptor (DOR1) are expressed in the dorsal root ganglia (DRG)of rats. In the present study, we determined the sizes of DRG neurons expressing DOR1 andMOR1 mRNAs and examined whether or not DRG neurons were likely to be the source of theDOR1 and MOR1 immunoreactivity previously observed in the spinal dorsal horn. DRG neuronswere labeled in five male Sprague-Dawley rats by applying Fluoro-Gold (FG) topically to thedorsal root entry zone. Five-micrometer cryostat sections were cut, and in situ hybridization wasperformed using full-length cRNA probes labeled with 35S-UTP. The distribution of sizes of DRGneuronal profiles (1372 neuronal profiles were evaluated) ranged from 98 to 2081 mm2 and wassimilar to those found in previous reports. Of 583 retrogradely labeled neuronal profiles in DRGs,246 (40 6 14%, mean 6 SD, n 5 5) expressed MOR1 mRNA. Of 789 DRG cell profiles fromsections that were hybridized for DOR1 mRNA, 687 (85 6 18%, mean 6 SD, n 5 5) were labeledfor DOR1. The proportion of DRG cell profiles expressing DOR1 mRNA was significantly higherthan that expressing MOR1 mRNA (P , 0.0001, chi-square test). No significant differences wereobserved between small (#700 mm2) and large (.700 mm2) FG-labeled neurons in the proportionslabeled for either MOR1 mRNA (202/497 vs. 44/86, P . 0.2, chi-square test) or DOR1 mRNA(555/651 vs. 132/138, P . 0.3, chi-square test). Most FG-labeled neurons that expressed eitherMOR1 mRNA or DOR1 mRNA (82.1 and 80.8%, respectively) were smaller than 700 mm2. Inaddition to cells expressing a single opioid receptor, individual DRG neurons were observed thatexpressed both MOR1 and DOR1. In a sample of 25 DRG neurons expressing MOR1-mRNA, 23also expressed DOR1 mRNA. Within the spinal cord itself, DOR1 and MOR1 mRNAs haddifferent patterns of expression. Both were expressed in the dorsal horn, but of the two, onlyMOR1 message was expressed in the superficial dorsal horn. We conclude that both small andlarge DRG neurons express DOR1 and MOR1 mRNAs, but most cells expressing these mRNAsare small. In addition, some DRG neurons express both MOR1 and DOR1 mRNAs. Finally, bothDOR1 and MOR1 in the spinal dorsal horn originate, at least in part, from DRG neurons. J.Comp. Neurol. 429:590–600, 2001. © 2001 Wiley-Liss, Inc.

Indexing terms: in situ hybridization; fluorescent dyes; neural pathways; cell counts; pain;

opiates

Previous studies have suggested that opioid-producedspinal analgesia requires both presynaptic mechanisms(involving inhibition of nociceptive primary afferent ter-minals) and postsynaptic mechanisms (involving inhibi-tion of spinal neurons; Duggan and North, 1984). Immu-noreactivities for both the cloned m-opioid receptor(MOR1) and the cloned d-opioid receptor (DOR1) havebeen shown in dorsal root ganglia (DRG) as well as in thespinal dorsal horn, wherein many primary afferent neu-rons terminate (Dado et al., 1993; Arvidsson et al.,1995a,b; Ji et al., 1995). In addition, in situ hybridizationstudies have also demonstrated that both MOR1 andDOR1 mRNAs exist in rat lumbar DRGs (Maekawa et al.,

1994; Mansour et al., 1994; Minami et al., 1995). Thesedata suggest that both MOR1 and DOR1 play roles in the

Grant sponsor: National Institute on Drug Abuse; Grant numbers PHSDA 05466, PHS DA 09642.

Hong Wang’s current address is: Department of Pharmacology, Univer-sity of Virginia, 1300 Jefferson Park Avenue, Charlottesville, VA 22908.

*Correspondence to: Martin W. Wessendorf, Ph.D., Department of Neu-roscience, University of Minnesota, Minneapolis, MN 55455.E-mail: [email protected]

Received 20 October 1999; Revised 26 September 2000; Accepted 5October 2000.

THE JOURNAL OF COMPARATIVE NEUROLOGY 429:590–600 (2001)

© 2001 WILEY-LISS, INC.

modulation of nociception at the level of the primary af-ferent terminal.

Nociception appears to be mediated largely by primaryafferent neurons with slow conduction velocities (Collinset al., 1960). DRG neurons with slow conduction velocities(and in particular, cells with conduction velocities in therange of C-fibers) have been reported to have cell bodiesthat are consistently smaller than those of faster conduct-ing neurons (Harper and Lawson, 1985); thus a tendencytoward small somata would be expected for nociceptiveneurons. Since there is relatively little evidence for directactions of opioids on non-nociceptive primary afferents(see, for instance, Seeber et al., 1978; Curtis et al., 1984;Hao et al., 1990), it would be expected that opioid recep-tors are expressed predominantly by small-diameter, no-ciceptive DRG neurons. There is evidence that this maynot be the case, however: Minami et al. (1995) describeexpression of opioid receptor mRNA by DRG neurons thatdo not express tachykinins (and that thus would be ex-pected to be non-nociceptive: see McCarthy and Lawson,1989; Lawson, 1995; Lawson et al., 1997). Moreover, non-quantitative studies report that opioid receptor mRNAsare expressed by at least some large-diameter DRG neu-rons (Mansour et al., 1994; Ji et al., 1995). However, it hasnot yet been determined whether the expression of opioidreceptors by large-diameter DRG neurons is common orrare.

In the present study, we examined the expression ofMOR1 and DOR1 mRNAs in the DRG cells of the lumbarspinal cord using quantitative in situ hybridization. Inparticular, we investigated the sizes of the DRG cellsexpressing either MOR1 or DOR1 mRNA. We addressedtwo questions: 1) do both small and large DRG cells ex-press MOR1 and/or DOR1 mRNA? and 2) if so, how com-mon is their expression of opioid receptor mRNAs?

MATERIALS AND METHODS

All procedures and protocols employing animals wereapproved by the University of Minnesota’s InstitutionalAnimal Care and Use Committee.

Retrograde labeling and preparationof tissue

Nissl staining of DRG neurons was severely compro-mised by the necessity to pretreat tissue with proteaseand RNase (see below). Therefore, to allow DRG neuronprofiles to be visible after sections were processed for insitu hybridization, neurons were retrogradely labeled us-ing hydroxystilbamidine isethionate (Fluoro-Gold [FG];Fluorochrome, Englewood, CO; Schmued and Fallon,1986; Wessendorf, 1991). Male Sprague-Dawley rats(100–200 g; Harlan, Madison, WI) were anesthetized byintramuscular injection of a mixture of ketamine (75 mg/kg), xylazine (5 mg/kg), and acepromazine (1 mg/kg). FGwas applied topically to the surface of the lumbosacralspinal cord as described previously (Kalyuzhny et al.,1996; Wang and Wessendorf, 1999). The skin was openedat the level of the 13th rib, a laminectomy was performed,and the dura was opened. Two microliters of a 5% solutionof FG in dimethylsulfoxide were absorbed into a piece ofgelfoam (;8 mm3) and placed on the dorsal root entryzone. The skin was closed, and rats were allowed to sur-vive 5–7 days before they were killed. FG has previously

been reported to be taken up avidly by the axons of pas-sage of primary afferents (Dado et al., 1990). Retrogradelylabeling cells had the added benefit of helping ensure thatonly neurons, as opposed to glia, were evaluated. In addi-tion, it confirmed that the neurons that were examinedprojected their axons to or through the dorsal spinal cord.

Rats were fixed by vascular perfusion (Wang and Wes-sendorf, 1999) after being anesthetized using the mixturedescribed above. Five hundred milliliters of fixative (4%paraformaldehyde and 14% (v/v) saturated picric acid in0.16 M phosphate buffer, pH 6.9) were followed by 400 mlof 10% sucrose solution in 0.1 M phosphate buffer, pH 7.2.After fixation, rats’ lumbar spinal cords and DRGs weredissected and placed in 10% sucrose solution overnight.The rats’ lumbar DRGs were randomly oriented onchucks, and 5-mm serial sections were cut using a Brightcryostat (Huntingdon, UK). Sections were stored at 220°Cuntil use.

In situ hybridization

The distributions of the mRNAs encoding MOR1 andDOR1 were examined in this study. Full-length cRNAprobes labeled with 35S-UTP were synthesized using thesame in vitro transcription approach as described previ-ously (Wang and Wessendorf, 1999). Full-length antisensesequences were used to localize the respective mRNAs;full-length sense sequences were used as controls.

In situ hybridization was also performed as describedpreviously (Wang and Wessendorf, 1999). Briefly, 5-mmcryostat sections were thawed onto silane-coated slides(Digene, Silver Spring, MD). Sections were deproteinatedwith 0.2 N HCl, delipidated with 0.005% digitonin, anddigested with 5 mg/ml proteinase K (Sigma, St. Louis, MO)at 37°C. The sections were then briefly dehydrated ingraded ethanols and acetylated using 0.1 M triethanol-amine and acetic anhydride (400:1, v/v). Sections wereagain dehydrated through graded ethanols and air-dried.Hybridization was performed at 45°C overnight with be-tween 105 and 6 3 105 cpm/section of labeled probe. Afterhybridization, sections were washed with 53 saline so-dium citrate buffer (SSC, pH 7.0) containing 10 mM di-thiothrietol (DTT) for 30 minutes at 42°C. A high-stringency wash was then performed for 20 minutes at60°C in a solution containing 50% formamide and 23 SSC.Unhybridized probe was digested with 25 mg/ml RNAse Aand 250 U/ml RNAse T1 at 37°C for 30 minutes in anRNAse digestion buffer consisting of 0.1 M Tris (pH 8.0),0.4 M NaCl, and 0.05 M EDTA. Slides were then washedsequentially in the RNAse digestion buffer, 23 SSC and0.13 SSC at 37°C, each for 15 minutes. Sections weredehydrated using 70% ethanol (containing 0.3 M NH4Ac tostabilize hybridized sequences), followed by 85% and100% ethanol, and the slides were allowed to dry at roomtemperature.

The slides were dipped in Kodak NTB2 and exposed at4°C. Exposure times were about 9 days for DOR1 andabout 21 days for MOR1. After exposure, the slides weredeveloped with Kodak Dektol Developer and fixed withKodak Fixer (Kodak, New Haven, CT), and the nuclei ofDRG cells were counterstained using 0.001% bisbenzim-ide (Hoechst 33258; Chemical Abstracts no. 23491-45-4;Schmued et al., 1982; Schnell and Wessendorf, 1995;Sigma, St. Louis, MO) in 0.2 M KCl/HCl, pH 7.2, for 2minutes. The sections were then rinsed in distilled water,

591SIZES OF DRG CELLS EXPRESSING OPIOID RECEPTORS

dehydrated through graded ethanols, and mounted withcoverslips.

Imaging

Microscopic images were collected on an Olympus BX50fluorescence microscope equipped with a 100-W Hg lamp.The filter sets allowed visualization of FG, bisbenzimidecounterstaining, and autoradiographic grains. Digital con-ventional microscopic images were collected using a Cohu4915 CCD camera (Cohu, San Diego, CA), a Power Macin-tosh 7100 computer equipped with a frame buffer (modelLG-3; Scion, Frederick, MD), and Scion’s version of thepublic domain National Institutes of Health (NIH) Imageprogram (developed at NIH and available from the Inter-net by anonymous FTP from zippy.nimh.nih.gov).

Digital images were manipulated with Adobe Photoshop(version 4.0.1; Adobe Systems, Mountain View, CA) andprinted with a Fuji Pictography 3000 (Tokyo, Japan) dig-ital color printer. Pairs of control and experimental im-ages (e.g., images of antisense labeling and sense controls)were manipulated identically.

Definition of an autoradiographicallylabeled neuron

Autoradiographically labeled cells were identified as de-scribed previously (Wang and Wessendorf, 1999). Briefly,an image of FG-labeled cells in a DRG was obtained usingfluorescence optics. The perimeter of a given FG-labeledcell was outlined, and its cross-sectional area was deter-mined. For consistency, cross-sectional areas were alwaysdetermined at the level of the cell nucleus. A brightfieldimage of the autoradiographic grains over the cell wasobtained and thresholded using standard illuminationconditions. In the autoradiographic image, the numbers ofabove-threshold pixels overlying the cell were counted; thenumbers of above-threshold pixels were also counted forthe entire microscopic field in which the cell was found.DRG neurons were considered autoradiographically la-beled if the density of pixels over them was significantlygreater than that of the microscopic field in which theywere observed (P , 0.0001, chi-square test). All measure-ments were made from images obtained with a 403,0.85 n.a. objective equipped with a correction collar forspherical aberration. Image analysis was performed usingNIH Image. Statistical comparisons were made using theStatView 4.5 software package (Abacus Concepts, Berke-ley, CA) or Prism 2.0 (GraphPad, San Diego, CA).

Quantification

As part of these studies, we determined the proportionsof DRG cells that were autoradiographically labeled foropioid receptor mRNA. Quantification was performed us-ing the DRGs from L4, -5, and -6. Because of the relativelack of independent landmarks (e.g., blood vessels, etc.) toaid identification of cells in sequential DRG sections, useof the physical disector was not practical, and profile-counting on single sections was used instead. To helpreduce stereological bias and to ensure that labeling wasnot evaluated at the very edge of a cell, profiles werecounted only if their nucleus was visible. Since large cellshave larger nuclei, the absolute numbers of large cellswould tend to be overestimated in these experiments.However, there would appear to be little effect on ourestimates of the relative proportions of mRNA-labeled and-unlabeled cells (see also Results and Discussion).

RESULTS

Injection sites

Application of FG onto the surface of the lumbosacralspinal cord resulted in labeling of the dorsal portion of thespinal cord in all five animals used in this study. A rep-resentative injection site is shown in Figure 1.

Frequency distribution of sizes ofFG-labeled DRG neuronal profiles

Spinal application of FG gave rise to a large number ofretrogradely labeled cells in DRGs (Fig. 2, FG). Most FG-labeled cells were small and had approximately ovaland/or circular shapes (Figs. 2, FG; 3, FG-BZ). Nuclei wereobserved in some FG-labeled neuronal profiles after coun-terstaining with 0.001% bisbenzimide (Figs. 2, BZ; 3, FG-BZ). The distribution of cross-sectional areas of DRG neu-ronal profiles ranged from 98 to 2081 mm2 (Fig. 4). Thedistribution of cross-sectional areas agrees with that re-ported in a previous study (Harper and Lawson, 1985),which suggested that C-fibers had somata with cross-sectional areas #700 mm2. Of 1372 neuronal profiles eval-uated, the cross-sectional areas of 1148 profiles (84%)were #700 mm2.

Sizes of DRG neuronal profilesautoradiographically labeled for MOR1 or

DOR1 mRNA

Many, but not all, DRG neuronal profiles were labeledfor MOR1 or DOR1 mRNA. The distribution of the sizes ofthese cell profiles appeared to be similar to that of thetotal population of FG-labeled DRG cells (see above). Thecross-sectional areas of DRG neuronal profiles rangedfrom 141 to 1875 mm2 in the case of MOR1 and from 98 to2081 mm2 in the case of DOR1 (Figs. 5, 6). Of 246 neuronalprofiles labeled for MOR1 mRNA, the sizes of 202 (82.1%)were #700 mm2. However, there was no significant differ-ence between the proportions of small (#700 mm2) andlarge (.700 mm2) cells that were labeled for MOR1 mRNA(202/497 vs. 44/86, P . 0.2, chi-square test). Of 687 DRGneuronal profiles autoradiographically labeled for DOR1mRNA, the sizes of 555 (80.8%) were #700 mm2. However,again there was no significant difference between the pro-portions of small (#700 mm2) and large (.700 mm2) cellsthat were labeled for DOR1 mRNA (555/651 vs. 132/138,P . 0.3, chi-square test).

Proportions of FG-labeled DRG neuronalprofiles autoradiographically labeled for

MOR1 and DOR1 mRNAs

The proportion of the total number of FG-labeled neu-ronal profiles that were autoradiographically labeled wasdetermined in the L4–6 DRGs using single-section profilecounts. It was found that 40 6 14% (mean 6 SD, n 5 5rats; 583 profiles evaluated) were autoradiographicallylabeled for MOR1 mRNA. Autoradiographic labeling forDOR1 appeared to be much more common; 85 6 18%(mean 6 SD; n 5 5 rats; 789 profiles evaluated) werelabeled for DOR1 mRNA. The proportion of DRG cellsexpressing DOR1 mRNA was significantly higher thanthat expressing MOR1 mRNA (P , 0.0001, chi-squaretest). There appeared to be no differences in the extent ofreceptor expression among segments L4–6. The propor-tions of DRG neuronal profiles that were autoradiographi-

592 H. WANG AND M.W. WESSENDORF

cally labeled for MOR1 mRNA were 43% (L4, 46/107), 41%(L5, 119/287), and 32% (L6, 29/92), respectively (n 5 3rats). There were no significant differences among theseproportions (P . 0.25, chi-square test). The proportions ofprofiles that were autoradiographically labeled for DOR1mRNA were 94% (L4, 218/232), 94% (L5, 286/305), and90% (L6, 123/137), respectively (n 5 3 rats). (These pro-portions appear to be higher than those listed above be-cause they were derived from a subpopulation of the totalnumber of rats.) Again, there were no significant differ-ences among these proportions (P . 0.25, chi-square test).

Co-expression of MOR1 and DOR1

Given the high proportion of DRG neurons expressingDOR1, it appeared likely that some DRG neurons labeledfor MOR1 also expressed DOR1. The latter appears to bethe case (Fig. 6). In a sample of MOR1-labeled neuronsidentified on pairs of semiadjacent 5-mm sections, 23 of 25were also labeled for DOR1.

Spinal cord

To determine the possible contribution of spinal neu-rons to the spinal expression of opioid receptors, we also

examined DOR1 and MOR1 mRNAs in lumbar spinalcord. As shown in Figure 7(MA), intense autoradiographiclabeling for MOR1 mRNA was observed in laminae I, II,IV, V, VI, VII, VIII, and X, but little labeling was seen inlaminae III and IX. Moderate autoradiographic labelingfor DOR1 mRNA was observed in laminae V, VI, VII, VIII,IX, and X, but only very weak labeling was observed in thesuperficial dorsal horn (laminae I and II) or in laminae IIIor IV.

Methodological controls

Figures 2 (MOR1-S and DOR1-S) and 7 (MS and DS)show the autoradiographic labeling obtained when at-tempting hybridization with sense strands (i.e., sequencesidentical to, rather than complementary to, the MOR1 orDOR1 mRNAs). Labeling resulting from use of sense se-quences for MOR1 and DOR1 mRNAs was diffuse andweak.

The proportions of DRG cells that were autoradiographi-cally labeled might have been over- or underestimated if thecells expressing receptor mRNA were larger or smaller thanthe population as a whole (Sterio, 1984). This appeared notto be the case. No significant differences were found between

Fig. 1. The extent of the Fluoro-Gold (FG) application site for one of the five rats used in these studies.Applications were made to the dorsal lumbosacral spinal cord. Images show the maximal extent in thetransverse plane of the spinal application site from the animal with the longest rostrocaudal extent of allapplication sites. Scale bar 5 400 mm.


Fig. 2. Low-magnification images of lumbar dorsal root ganglia(DRG) sections showing Fluorogold (FG) labeling, counterstaining ofnuclei by bisbenzimide (BZ), and autoradiographic labeling for thecloned m-opioid receptor (MOR1) mRNA and cloned d-opioid receptor(DOR1) mRNA. FG: Section contains cells retrogradely labeled by FG.Only neuronal somata are labeled. BZ: Section counterstained withBZ. Intense labeling of many glial nuclei is evident. In addition, thenuclei of some, but not all neurons are in the plane of section and are

labeled. MOR1-A, DOR1-A: Specific labeling for MOR1 and DOR1mRNAs, respectively, obtained using antisense probes. Labeling forMOR1 is less common, but more intense, than that for DOR1.MOR1-S, DOR1-S: Sense-sequence controls for MOR1 and DOR1,respectively, showing nonspecific labeling. The section shown inMOR1-S was adjacent to that in MOR1-A; similarly, the section inDOR1-S was adjacent to that shown in DOR1-A. Scale bar 5 200 mm.


MOR1-labeled and unlabeled cells in the cross-sectional areaof either the cell soma (P . 0.39, unpaired t-test) or the cellnucleus (P . 0.63, unpaired t-test), nor were any such dif-ferences observed between DOR1-labeled and -unlabeledcells in the cross-sectional areas of either the cell soma (P .0.23, unpaired t-test) or nucleus (P . 0.13, unpaired t-test;Tables 1 and 2).

DISCUSSION

There are three principal findings from these studies.First, most cells expressing MOR1 and DOR1 mRNAs aresmall. Second, equal proportions of small and large DRGneurons express MOR1 and DOR1 mRNAs. Third, someDRG neurons express both MOR1 and DOR1 mRNAs.

Stereological considerations

Single-section profile counting was employed for quan-tification in this study. Since cells that are larger in thez-axis are more likely to be sampled by this method (Ste-rio, 1984), the proportions of autoradiographically labeled

cells that we estimated could have been over- or underes-timated if the sizes of the cells (or of their nuclei, sinceonly cells with nuclei were counted) being measured weredifferent. We found no significant differences between au-toradiographically labeled and unlabeled cells in thecross-sectional areas of either their cell somata or theirnuclei; this was true for both MOR1-labeled cells andDOR1-labeled cells. Given the apparently spherical shapeof the nuclei of DRG neurons and the fact that DRGs weresectioned in a variety of orientations, it appears likely thatthe proportions we calculated were accurate.

Distribution of sizes of DRG neuronalprofiles expressing MOR1 and DOR1 mRNAs

In the present study, it was found that the distributionof the cross-sectional areas of DRG neuronal profiles ex-tended from about 100 to about 2000 mm2 but that mostDRG cells (over four-fifths of the cells) were small (i.e.,#700 mm2). This distribution pattern was similar to thosereported previously (for review, see Lawson, 1995).

Fig. 3. Two sets of images of single 5-mm sections containingFluorogold (FG)-labeled DRG cells that were counterstained by bis-benzimide (BZ) and hybridized with antisense probes either form-opioid receptor (MOR1) mRNA (left) or d-opioid receptor (DOR1)mRNA (right). Again, many neurons were labeled for either MOR1 orDOR1 mRNA. Left: Closed arrow points to a large (1134-mm2) DRGneuron with a nucleus counterstained by bisbenzimide that was la-beled for MOR1 mRNA. Open arrow points to a small neuron (259

mm2) autoradiographically labeled for MOR1 mRNA. Right: Closedarrow points to a large (2035-mm2) DRG neuron with a nucleus coun-terstained by bisbenzimide that was labeled for DOR1 mRNA. Openarrow points to a small neuron (367 mm2) autoradiographically la-beled for DOR1 mRNA. FG-BZ: FG labeling and bisbenzimide coun-terstaining. MOR1: Labeling for MOR1 mRNA in the same field asthe FG-BZ image. Scale bar 5 25 mm.


Consistent with previous studies (Maekawa et al., 1994;Mansour et al., 1994; Minami et al., 1995), autoradio-graphic labeling for MOR1 and DOR1 mRNAs was foundin lumbar DRGs. Our proportion of DRG neurons labeledfor MOR1 (40%) was somewhat lower than that reportedin the work of Minami et al. (1995; 60%). Conceivably thismay be due to differences in the probes used. In contrast,the proportion of DRG neurons that we found labeled forDOR1 mRNA (85%) was far higher than that reported byMinami et al. (1995; 37%). This again may be due todifferences in the probe used. However, it may more likelybe due to greater sensitivity of the statistical method weused for identifying labeled cells. Minami et al. (1995)used a flat criterion of 15 grains/cell, regardless of the cellsize. In our study, we used a statistical criterion (i.e., asignificantly higher density of labeling), which may haveallowed us to detect more neurons with relatively lightlabeling. In agreement with our findings, it has previouslybeen reported that many DOR1-labeled neurons arelightly labeled (Minami et al., 1995).

In our study, the distributions of the sizes of profilesautoradiographically labeled for either MOR1 or DOR1mRNA appeared to be similar to the general pattern ofdistribution of DRG neuronal profiles. As mentionedabove, no significant differences were observed betweenthe proportions of small and large DRG cells labeled forMOR1 mRNA, nor was any difference noted for DOR1mRNA. Thus we conclude that these opioid receptors arenot preferentially expressed by small-diameter primaryafferent neurons. However, previous studies have re-ported DOR1 immunoreactivity in a pattern consistentwith the distribution of small-diameter primary afferentsrather than large-diameter primary afferents (Dado et al.,1993; Arvidsson et al., 1995a; Ji et al., 1995). This discrep-ancy suggests at least three interpretations. First, it ispossible that less DOR1 is translated into protein in large-diameter primary afferent neurons, i.e., that in situ hy-bridization is more sensitive at detecting DOR1-expressing neurons than immunohistochemistry. Second,it is possible that the distribution of large-diameter pri-

mary afferent endings in the spinal cord is not as easy torecognize as the distinctive pattern of small-diameter af-ferents in the superficial dorsal horn. Third, it is possiblethat large-diameter DOR1-immunoreactive axons arepresent but have not been recognized due to DOR1 beingpresent mainly in terminal portions. At present it is notpossible to distinguish between these possibilities.

Previous studies have illustrated coexpression of MOR1and DOR1 in axon varicosities in the superficial dorsalhorn (Arvidsson et al., 1995b; Ji et al., 1995), suggestingthat DOR1 and MOR1 may be co-expressed in DRG neu-rons. In the present study we found direct evidence forco-expression of MOR1 and DOR1 at the level of themRNA (Fig. 6). Moreover, co-expression of DOR1 mRNAappears to be very common in MOR1-expressing cells. Thepresence of DOR1 and MOR1 mRNAs in single neuronshas previously been reported in brainstem (Wang andWessendorf, 1999). Co-expression of these receptors sug-

Fig. 4. Histogram showing the frequency distribution of the cross-sectional area of lumbar DRG neuronal profiles. Both small (#700mm2) and large (.700 mm2) neurons were observed.

Fig. 5. Histogram showing the frequency distribution of the cross-sectional areas of lumbar DRG neuronal profiles that were autoradio-graphically labeled for m-opioid receptor (MOR1) mRNA (A) ord-opioid receptor (DOR1) mRNA (B). The open bars give the totalnumber of profiles counted; the filled bars show the number of profileslabeled for MOR1 mRNA. The proportion of profiles that were labeledfor either DOR1 or MOR1 was found not to differ between small cells(#700 mm2) and large cells (.700 mm2).


Fig. 6. The mRNAs encoding the cloned d-opioid receptor (DOR1)and the cloned m-opioid receptor (MOR1) coexist in single DRG neu-rons. Plate shows images from two 5-mm sections of lumbar DRG thatwere separated by 5 mm. Left: Section hybridized for DOR1. Right:Images of the section hybridized for MOR1. A,B: FG labeling in thetwo sections. Neurons marked by numbers appear to be visible in bothsections. C,D: Images showing locations of autoradiographic grainsrelative to the FG-labeled cells. FG profiles are outlined on the im-

ages. E,F: Graphs showing density of autoradiographic labeling overFG-labeled cells. BG, background level of labeling. Densities of label-ing were compared using a chi-square test (see Materials and Meth-ods). Cells 1, 2, and 3 were labeled for both MOR1 and DOR1 atdensities significantly higher than background (chi-square test, P ,0.0001). Cell 4 was labeled for DOR1 but not for MOR1. Scale bar 525 mm.


gests that different types of opioid ligands (i.e., both m andd) may be able to affect the same cell. In addition, coex-pression of these receptors is consistent with the presenceof m-d receptor dimerization (Gomes et al., 2000). If dimer-ization is occurring, it may indicate that the pharmacologyof these receptors is not the same as that of “classical”m- and d-opioid receptors (Gomes et al., 2000).

m-Opioid receptors and primaryafferent neurons

In this study, over one-third of FG-labeled lumbar DRGneuronal profiles were found to express MOR1 mRNA.(Alternatively spliced forms of the cloned m-opioid receptorhave recently been described [Bare et al., 1994; Zimprichet al., 1995; Pan et al., 1999; Abbadie et al., 2000], and itappears that at least some of these are expressed by pri-mary afferent neurons as well.) MOR1 immunoreactivityhas previously been shown in DRG neurons and in sites oftheir central termination in the superficial dorsal horn(Arvidsson et al., 1995b; Ji et al., 1995). In addition, dorsalrhizotomy has been reported to reduce m-opioid ligandbinding (see, for instance, Stevens and Seybold, 1995) andMOR1 immunoreactivity (Arvidsson et al., 1995b). Thesefindings suggest that m-opioid agonists act, at least inpart, directly on primary afferent terminals. They are also

consistent with studies reporting that m-opioid agonistshave direct effects on primary afferent neurons (Werz etal., 1987; Taddese et al., 1995; although see also Shefneret al., 1981; Williams and Zieglgansberger, 1981). MOR1immunoreactivity has also been observed in large-diameter DRG neurons and in the deeper portions of thedorsal horn (Arvidsson et al., 1995b; Ji et al., 1995),wherein large-diameter primary afferents terminate (Wil-lis and Coggeshall, 1991). The latter findings are consis-tent with our observations that large-diameter DRG neu-rons also express MOR1 mRNA.

In addition, strong autoradiographic labeling for MOR1mRNA was found in the superficial dorsal horn of thespinal cord (Fig. 7,MA). This is consistent with previousstudies (Maekawa et al., 1994; Mansour et al., 1994) andsuggests that m-opioid agonists may modulate antinoci-ception by means of both spinally projecting DRG neuronsand neurons intrinsic to the spinal cord.

d-Opioid receptors and primaryafferent neurons

Immunoreactivity for DOR1 has previously been re-ported in sensory neurons (Dado et al., 1993; Arvidsson etal., 1995a; Ji et al., 1995), and DOR1-immunoreactivefibers and terminals in the superficial dorsal horn have

Fig. 7. Adjacent sections of L5 spinal cord, hybridized for MOR1 (above) or DOR1 (bottom) either withantisense probes or sense (control) sequences. MA: Labeling for MOR1 mRNA. MS: MOR1 sense probe.DA: labeling for DOR1 mRNA. DS: DOR1 sense probe. Labeling for MOR1 (MA) but not DOR1 (DA) canbe observed in the superficial dorsal horn.


been reported to decrease after dorsal rhizotomy (Dado etal., 1993). In the present study, we found that over five-sixths of lumbar DRG neurons expressed DOR1 mRNA,suggesting that d-opioid agonists can directly affect pri-mary afferent neurons. The latter conclusion is consistentwith electrophysiological studies reporting effects ofd-opioid agonists on spinal afferent input (Glaum et al.,1994) and on calcium currents in DRG neurons (Acostaand Lopez, 1999).

In the spinal cord, DOR1 mRNA was observed to bemainly expressed in laminae V, VI, VII, VIII, IX, and X,with little specific labeling in the superficial dorsal hornand the neck of the dorsal horn in the lumbar spinal cord(Fig. 7, DA; see also Mansour et al., 1994). This contrastsstrikingly with the dense DOR1-immunoreactivity in thesuperficial dorsal horn (Dado et al., 1993; Arvidsson et al.,1995a; Ji et al., 1995). These results suggest that DOR1 isaxonally transported to the superficial dorsal horn fromother sites, e.g., primary afferent neurons, spinal neuronsoutside the superficial dorsal horn, or bulbospinal neurons(Wang and Wessendorf, 1999). d-Opioid receptors havebeen reported to inhibit the transmission of nociceptiveneural activities in the superficial dorsal spinal cord pre-synaptically (Glaum et al., 1994). Our findings are consis-tent with that conclusion.

Physiological significance

Large-diameter, rapidly conducting primary afferentneurons appear to transmit innocuous sensation (Collinset al., 1960). Conversely, pain appears to be transmittedby small-diameter, slowly conducting primary afferents.Excitation of the smallest, slowest conducting fibers,C-fibers, is associated with an extremely aversive sensa-tion of burning pain (Collins et al., 1960). In rats, it hasbeen reported that most DRG neurons with cross-sectionalareas #700 mm2 had axons conducting in the A-d orC-fiber range. Moreover, all cells that had axons conduct-ing in the C-fiber range (i.e., ,2 m/sec) had somata mea-suring less than 700 mm2 (Harper and Lawson, 1985). Inaddition, these small DRG neurons express substance P(McCarthy and Lawson, 1989; Lawson et al., 1997) andare nociceptive (Lawson et al., 1997). Conversely, mostcells with somata larger than 700 mm2 had conductionvelocities greater than 10 m/sec (the approximate upperlimit for A-d fibers; Harper and Lawson, 1985). Theselarge DRG neurons appear only rarely to express sub-stance P (Lawson et al., 1997) and appear to be predomi-

nantly non-nociceptive (Collins et al., 1960; Ertekin et al.,1975). Thus, although it is not an absolute line of demar-cation (Harper and Lawson, 1985), a somatic cross-sectional area of 700 mm2 appears to mark a useful divi-sion between slower and faster conducting DRG neurons.

The expression of opioid receptors on small-diameterprimary afferent neurons is consistent with opioids pre-synaptically inhibiting nociception, at least in part, at thelevel of the primary afferent terminal (Yaksh, 1977; al-though see also Suarez-Roca and Maixner, 1993). It is alsoconsistent with previous studies indicating that small-diameter primary afferent terminals express d- andm-opioid receptors (LaMotte et al., 1976; Zajac et al., 1989;Gouarderes et al., 1991; Dado et al., 1993; Arvidsson et al.,1995b; Stevens and Seybold, 1995). However, our datasuggest that opioid receptors are present to an equal ex-tent in large DRG neurons that are in all likelihood non-nociceptive. Consistent with our findings, a previous dou-ble in situ hybridization study (Minami et al., 1995)suggested that both m- and d-opioid receptor mRNAs wereexpressed in DRG neurons, including some DRG neuronsthat did not express substance P. (Substance P appearsnot to be expressed by large-diameter primary afferents;Lawson, 1995.)

Although small-diameter primary afferent neurons ter-minate in laminae I and II, laminae III and IV appear toreceive predominantly large, non-nociceptive primary af-ferent terminals (Willis and Coggeshall, 1991). Previousstudies have reported that m-opioid binding sites in spinallaminae III and IV (as well as in laminae I and II) aredecreased after dorsal rhizotomy (Gouarderes et al.,1991). Our findings of m-opioid receptors on large-diameter DRG neurons are consistent with these studies.

Opioid receptors are thought to inhibit neurotransmis-sion from small-diameter nociceptive primary afferents.Thus it might be expected that activation of opioid recep-tors on large-diameter, non-nociceptive primary afferentsmight also be inhibitory. It has been suggested that en-dogenous opioids decrease the excitability of non-nociceptive primary afferents (Duggan et al., 1985). It hasalso been reported that activation of d-opioid receptorsinhibits non-nociceptive spinal reflexes (Schmidt et al.,1991). Although such effects are not always observed(Curtis et al., 1984; Willer et al., 1988; Hao et al., 1990), itappears possible that activation of opioid receptors servesto inhibit non-nociceptive primary afferents as well asnociceptive primary afferents. Opioid inhibition of non-

TABLE 1. Cross-Sectional Areas (mm2; mean 6 SEM) of Somatic Profiles of DRG Neurons1

mRNA

Large cells Small cells

Labeled Unlabeled Labeled Unlabeled

MOR1 1062 6 41.3 (n5 44) 1013 6 41.3 (n542) 360.6 6 9.4 (n5202) 359.5 6 7.3 (n5295)DOR1 1114 6 26.4 (n5132) 1069 6 117.2 (n5 6) 362.1 6 6.0 (n5555) 343.8 6 12.4 (n5 96)

1No significant differences were seen in somatic cross-sectional area between autoradiographically labeled and unlabeled cells (P . 0.23, unpaired t-test).

TABLE 2. Cross-Sectional Areas (mm2; Mean 6 SEM) of Nuclei of DRG Neurons1

mRNA

Large cells Small cells

Labeled Unlabeled Labeled Unlabeled

MOR1 122.1 6 6.2 (n5 44) 124.4 6 6.0 (n542) 76.1 6 2.4 (n5202) 77.6 6 1.9 (n5295)DOR1 126.2 6 3.3 (n5132) 102.9 6 18.4 (n5 6) 70.5 6 1.4 (n5555) 75.7 6 3.2 (n5 96)

1No significant differences were seen in nuclear cross-sectional area between autoradiographically labeled and unlabeled cells (P . 0.13, unpaired t-test).


nociceptive primary afferents has been suggested to de-crease movement after injury (Duggan et al., 1984).

In conclusion, MOR1 and DOR1 mRNAs are expressed byequal proportions of small and large DRG neurons, althoughmost of the cells expressing MOR1 or DOR1 mRNA aresmall. The precise roles of DOR1 and MOR1 in large-diameter non-nociceptive primary afferents and the co-existence of DOR1 with MOR1 remain to be further clarified.

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Equal proportions of small and large DRG neurons express opioid receptor mRNAs

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