participation of μ-opioid, gabab, and nk1 receptors of major pain control medullary areas in...

Participation of �-Opioid, GABAB, andNK1 Receptors of Major Pain Control

Medullary Areas in Pathways Targetingthe Rat Spinal Cord: Implications for

Descending Modulation ofNociceptive Transmission

MARTA PINTO,1-3 MARTA SOUSA,1,2 DEOLINDA LIMA,2,3AND ISAURA TAVARES1,2*

1Institute of Histology and Embryology, Faculdade de Medicina, Universidade do Porto,4200-319 Porto, Portugal

2Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto,4150-180 Porto, Portugal

3Laboratory of Molecular Cell Biology, Faculdade de Medicina, Universidade do Porto,4200-319 Porto, Portugal

ABSTRACTSeveral brain areas modulate pain transmission through direct projections to the spinal cord.

The descending modulation is exerted by neurotransmitters acting both at spinally projectingneurons and at interneurons that target the projection neurons. We analyzed the expression of�-opioid (MOR), �-aminobutyric acid GABAB, and NK1 receptors in spinally projecting neuronsof major medullary pain control areas of the rat: rostroventromedial medulla (RVM), dorsalreticular nucleus (DRt), nucleus of the solitary tract, ventral reticular nucleus, and lateralmostpart of the caudal ventrolateral medulla. The retrograde tracer cholera toxin subunit B (CTb) wasinjected into the spinal dorsal horn, and medullary sections were processed by double immuno-cytochemistry for CTb and each receptor. The RVM contained the majority of double-labeledneurons followed by the DRt. In general, high percentages of MOR- and NK1-expressing neuronswere retrogradely labeled, whereas GABAB receptors were mainly expressed in neurons thatwere not labeled from the cord. The results suggest that MOR and NK1 receptors play animportant role in direct and indirect control of descending modulation. The co-localization ofMOR and GABAB in DRt neurons also demonstrated by the present study suggests that thepronociceptive effects of this nucleus may be controlled by local opoidergic and GABAergicinhibition of the pronociception increased during chronic pain. J. Comp. Neurol. 510:175–187,2008. © 2008 Wiley-Liss, Inc.

Indexing terms: opioid receptors; substance P; dorsal reticular nucleus; endogenous pain control

system; antinociception; pronociception

Nociceptive transmission at the spinal dorsal horn ismodulated from several supraspinal areas. The medullaoblongata is the brain area with the higher density of painmodulatory centers, including the rostroventromedial me-dulla (RVM), the dorsal reticular nucleus (DRt), the nu-cleus of the solitary tract (Sol), the ventral reticular nu-cleus (VRt), and the caudal ventrolateral medulla (VLM)(reviewed by Jones, 1992; Stamford, 1995; Millan, 2002).Anatomical studies revealed projections to the spinal dor-sal horn from the RVM (Martin et al., 1985), DRt (Almeidaet al., 1993; Villanueva et al., 1995), Sol (Mtui et al., 1993),VRt (Tavares and Lima, 1994), and VLM (Tavares and

Lima, 1994; Tavares et al., 1996). The functional rele-vance of these findings was demonstrated. For example,

Grant sponsor: Bial Foundation; Grant number: 15/04; Grant sponsor:Fundacao para a Ciencia e a Tecnologia (FCT) (Portugal) PhD grant (to M.P.).

*Correspondence to: Isaura Tavares, Institute of Histology and Embry-ology, Faculty of Medicine of Oporto, Alameda Hernani Monteiro, 4200-319Porto, Portugal. E-mail: [email protected]

Received 3 July 2007; Revised 18 January 2008; Accepted 28 May 2008DOI 10.1002/cne.21793Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 510:175–187 (2008)

© 2008 WILEY-LISS, INC.

stimulation of the Sol and VRt induces analgesia anddepresses nociceptive responses of spinal neurons (Aicherand Randich, 1990). Spinally projecting VLM neurons arelocated at its lateralmost portion, designated VLMlat (Ta-vares and Lima, 1994; reviewed by Tavares and Lima,2002), and this is the VLM region from which analgesia ismore effectively induced upon local stimulation (Gebhartand Ossipov, 1986; Janss and Gebhart, 1988). The antino-ciceptive effects triggered from the medulla oblongata co-exist with pronociceptive actions.

The RVM, which plays a pivot role in conveying to thespinal cord the antinociceptive effects elicited from theperiaqueductal grey (PAG), exerts both inhibitory andfacilitatory effects. It has been proposed that inhibitionand facilitation are triggered by subpopulations of RVMneurons, respectively, “OFF-cells” and “ON-cells” (re-viewed by Heinricher et al., 2003; Gebhart, 2004). TheDRt plays a peculiar role in descending modulation be-cause it is exclusively devoted to descending facilitation(reviewed by Lima and Almeida, 2002). Stimulation of theDRt induces hyperalgesia in acute and sustained painmodels (Almeida et al., 1996, 1999) and increases thenociceptive responses of spinal neurons (Dugast et al.,2003). It was recently demonstrated that descending fa-cilitation from the DRt contributes to maintenance ofchronic pain states (Sotgiu et al., 2008).

Opioids and �-aminobutyric acid (GABA) provide themain neurochemical substrate for local modulation of thepain control areas referred above. Most data have beencollected for the RVM due to its pivotal role in the well-established PAG-triggered analgesia. Spinally projectingRVM neurons expressing opioid receptors appear to me-diate the opioid analgesia triggered from the PAG (Kiefelet al., 1993; Kalyuznhy et al., 1996; Hirakawa et al., 1999;Vasquez and Vanegas, 2000), and microinjection of theMOR agonist (D-ALA2,N-ME-PHE4,GLY5-OL)-enkephalinacetate (DAMGO) into the RVM elicits analgesia (Fields etal., 1983; Rossi et al., 1994; Hurley et al., 2003). As to theGABAergic system, spinally projecting RVM neurons ex-press GABAB receptors (Yang et al., 2002), and microin-jection of the GABAB agonist baclofen into the RVM in-duces analgesia (Drower and Hammond 1988; Thomas etal., 1995). Regarding the substance P (SP)ergic system,recent studies indicate that SP, acting upon NK1 recep-

tors, might also be relevant in descending pain control.NK1 knockout mice have substantial impairments of en-dogenous pain control mechanisms (Bester et al., 2001).

The actions of opioids and GABA in descending modu-lation appear to be strongly related. Co-expression ofMOR and GABAB receptors was demonstrated in severalareas of the supraspinal pain control system (Murakamiet al., 1987; Li et al., 1996, 1997; Kalyuzhny et al., 1998).The activation of MOR is known to modulate GABAergicneurons in pain control centers (Kalyuzhny et al., 1998),and the analgesia induced by microinjection of opioid ago-nists into the RVM is blocked by GABAB antagonists(Hurley et al., 2003).

We previously demonstrated that neurons of the RVM,DRt, Sol, VRt, and VLMlat, upon c-fos activation in re-sponse to noxious stimulation, strongly express MOR andGABAB receptors (Pinto et al., 2003). In order to under-stand the involvement of MOR, GABAB, and NK1 recep-tors in descending pain control, the present study charac-terizes the expression of those receptors in bulbospinalpathways originated at the aforementioned medullary ar-eas. Immunocytochemistry for each receptor was com-bined with detection of the tracer cholera toxin subunit B(CTb) retrogradely transported from the spinal dorsalhorn. Due to the special role of the DRt in pronociceptivemodulation and the interaction between opioidergic andGABAergic modulation, we also studied the co-expressionof MOR and GABAB receptors at the DRt.

MATERIALS AND METHODS

All experiments were carried out in accordance with theEuropean Community Council Directive (86/609/EEC)and ethical guidelines for pain investigation in animals(Zimmermann, 1983).

Expression of MOR, GABAB, and NK1 inspinally projecting neurons

CTb injections at the spinal dorsal horn. Adultmale Wistar rats, 320–340 g in weight, derived from thecolony of Charles River (Barcelona, Spain), were housedunder temperature- and light-controlled conditions (22°C;lights on between 8 a.m. and 8 p.m.), with free access tofood and water. Twelve animals were intraperitoneally(ip) anesthetized with a mixture of medetomidine (0.25mg/kg body weight) and ketamine (60 mg/kg body weight)and subjected to a dorsal laminectomy of spinal vertebraeC4–C7. The dura mater was slit, and four pressure injec-tions, 0.25 �l each, of 1.5% CTb (List Biological Laborato-ries, Campbell, CA) were bilaterally performed about 1mm lateral to the dorsal vertebral artery, as previouslydescribed (Tavares and Lima, 1994).

Four days after CTb injections, the animals were anes-thetized with sodium pentobarbital ip (50 mg/kg bodyweight) and perfused through the ascending aorta with100 ml of calcium-free Tyrode’s solution, followed by 750ml of a fixative solution containing 4% paraformaldehydeand 14% picric acid in 0.1 M phosphate buffer (PB), pH6.9. The brainstem and spinal cord were removed, post-fixed by immersion for 3 hours in fixative, and cryopro-tected with 30% sucrose solution in 0.1 M phosphate-buffered saline (PBS), overnight, at 4°C. Coronal frozensections, 40 �m thick, obtained from the C4–C7 spinalsegments and from the medulla oblongata, were serially

Abbreviations

Cu cuneate nucleuscc central canalCTb cholera toxin subunit BDAMGO (D-ALA2,N-ME-PHE4,GLY5-OL)-enkephalin acetateDRt dorsal reticular nucleusGAD glutamic acid decarboxylaseGABA �-aminobutyric acidicv intracerebroventricularip intraperitoneallyIR immunoreactiveLRt lateral reticular nucleusMOR �-opioid receptorsPAG periaqueductal graySol nucleus of the solitary tractRVM rostroventromedial medullaSP substance PVLM caudal ventrolateral medullaVLMlat lateral reticular formation of the caudal ventrolateral me-

dullaVRt ventral reticular nucleus

The Journal of Comparative Neurology

176 M. PINTO ET AL.

collected in PBS. Spinal sections were processed by CTbimmunostaining to determine the location of the injectionsites. The rostrocaudal order of the medullary sectionswas maintained, and sections were alternately processedin double immunoreactions for CTb and each receptor (seedouble immunodetection section below). The final num-bers of sections used in the counting in each medullaryarea and animal experiment are given in Tables 1 and 2.An average of 21 sections was analyzed in each experi-ment, with about 17 sections encompassing the RVM and4 sections from the remaining medullary nuclei.

Antibodies. The polyclonal anti-CTb antibody (ListBiological Laboratories; Cat No 703, Lot No GAC-O1C)was raised in goat. A control experiment was performed toconfirm the antibody specificity by using medullary sec-tions of an animal not injected with CTb and perfused asdescribed above. The lack of labeling confirmed the reli-ability of the CTb antibody, in accordance with previousreports (Gritti et al., 1997; Coolen and Wood, 1998; Kol-mac et al., 1998).

The antibody against MOR (Neuromics; Cat NoRA10104, Lot No 400058) was raised in rabbit, and theimmunogen sequence corresponded to residues 1359–1403 of the C-terminus of MOR (NHQLENLEAETAPLP).Other studies using antibodies against similar peptidesequences showed a distribution pattern of MOR immu-nostaining (Mansour et al., 1995; Arvidsson et al., 1995)identical to that observed in the present study. We testedthe antibody specificity further by blocking the antibodywith the MOR blocking peptide (Neuromics, P10104) bothin immunocytochemistry and in Western blot analysis.The general procedures of an immunocytochemistry reac-tion are described in detail in the following sections. Inbrief, sections of an additional animal obtained as de-scribed before were incubated with MOR antibody or withMOR antibody previously blocked with 1 �g/ml of blockingpeptide, for 1 hour. Labeling at the medulla oblongata(Fig. 1A) was abolished when the primary antibody wasblocked by preabsorption (Fig. 1B), supporting the speci-ficity of the anti-MOR antibody. For the Western blotanalysis, two additional animals were used.

The spinal cord and brainstem were removed, washed inPBS, and transferred into a lysate buffer (Tris-HCl 0.05M, pH 7.4, 0.32 M sucrose, and a cocktail of proteaseinhibitors from Sigma, St. Louis, MO; P8340). Seventymicrograms of the proteins obtained were resolved on a12% sodium dodecyl sulfate-polyacrylamide gel and blot-ted onto a nitrocellulose membrane. The membrane wasblocked with TBS 0.05 M, pH 7.2, containing 5% driedmilk and 0.05% Tween 20, for 1 hour, and incubated fortwo overnights at 4°C with anti-MOR (at 1:3,000) or withanti-MOR previously incubated for 1 hour with 1 �g/ml ofMOR blocking peptide. The detection was performed withthe ECL chemoluminescent method following the manu-facturer’s instructions (Amersham Bioscience, Bucking-hamshire, UK). A single band was observed in the spinalcord (SC) and brainstem (BS) material (Fig. 1C) with 70kDA, which matches the molecular weight of MOR (Kivellet al., 2004).

The antibody against GABAB receptors (Chemicon, Te-mecular, CA; Cat No AB1531, Lot No 23071241) was pro-duced in guinea pig against a sequence (PSEPPDRLSC-DGSRVHLLYK) common to both GABAB R1a and GABABR1b receptors subunits of the rat. In Western blots ofspinal cord tissue, this antibody was previously shown to

stain a band at 117 kDa (Castro et al., 2006a), supportingthe specificity of this antibody (Fillipov et al., 2000). Lossof staining with this GABAB antibody was previouslyshown in the brains of GABAB knockout mice (Gassmannet al., 2004). In the present study we performed preab-sorption control experiments for the GABAB antibody byusing similar methods to those described for the anti-MOR. The reactivity of the GABAB antibody was com-pletely blocked by 1 �g/ml of GABAB receptor R1 controlpeptide (Chemicon, AG324) in immunocytochemical andWestern blot analysis. The 117-kDa band detected afterincubation with the anti-GABAB receptor, at a 1:500 dilu-tion, for 40–42 hours was absent when the antibody waspreviously blocked by preadsorption.

The antibody against NK1 (Chemicon; Cat No AB5060,Lot No 23030368) was a rabbit polyclonal antibodyagainst a peptide sequence (KTMTESFSFSSNVLS) corre-sponding to residues 393–407 of the C-terminus (Vigna etal., 1994). The specificity of this antibody was demon-strated in the retina by absence of staining in knockoutmice (Catalani et al., 2006). After selective lesion of NK1-expressing neurons with substance P conjugated with sa-porin, a significant decrease in NK1 staining was detectedin the spinal cord (Manthy et al., 1997; Castro et al.,2006b) and in the brain (Saka et al., 2002), supporting itsspecificity. The specificity of this antibody was also testedby preadsorption control experiments (Casini et al., 1997,2004; Piggins et al., 2001), Western blot analysis (Zhanget al., 2007), and omission of the primary antibody (Zhanget al., 2007).

Fig. 1. A,B: Photomicrographs of medullary sections immunore-acted with the anti-MOR antibody (MOR in A) or with the anti-MORantibody blocked by preadsorption (MOR�block in B). C: Westernblot analysis of spinal cord (SC) and brainstem (Br) material immu-noreacted to MOR. A 70-kDa band is shown, in agreement withprevious studies (Kivell et al., 2004). The band is absent when theMOR antiserum is preabsorbed (MOR�block). Scale bar � 50 �m inB (applies to A,B).


177BULBOSPINAL NEURON RECEPTORS AND PAIN CONTROL

The anti-glutamic acid decarboxylase (GAD; Develop-mental Studies Hybridoma Bank, Iowa City, IA; GAD-6)was a monoclonal antibody against the GAD antigen fromrat brain. The specificity of this antibody was demon-strated by Western blot analysis (Chang and Gottlieb,1988; Jevince et al., 2006).

The specificity of the secondary antisera used in eachimmunoreaction was demonstrated by omitting the incu-bations in the respective primary antibodies.

Single CTb immunoreaction. One in every three spi-nal sections were incubated for 48 hours at 4°C in the goatantiserum against CTb referred to above (List BiologicalLaboratories), at a 1:4,000 dilution, in a 0.1 M PBS solu-tion containing 0.3% Triton X-100 (PBST). Following re-peated washes, the sections were incubated in a biotinyl-ated horse anti-goat antibody (Vector, Burlingame, CA),followed by an ABC solution (Vectorstain Elite, Vector),both for 1 hour at a 1:200 dilution. The DAB chromogenwas used as described previously (Tavares et al., 1997),and the sections were mounted on gelatin-coated slides,cleared in xylol, and coverslipped with Eukitt. Only ani-mals with injection sites restricted to the dorsal horn wereincluded in the study (n � 8).

Double immunodetection of CTb plus MOR, GABAB,

or NK1 receptors. Three in every four medullary sec-tions were processed by using double-immunocytochemicalreactions for CTb and each receptor. The fourth set ofsections was used to test the specificity of the antibodies inthe preadsorption control experiments referred to above.In all the double immunocytochemical reactions, incuba-tions and washings were performed in the PBST solutionused above. In order to decrease background staining, theincubation in each primary antiserum was preceded byimmersion, for 2 hours, in PBST containing 0.1 M glycineand 10% normal serum raised in the animal species fromwhich the respective secondary antibodies were produced.Sections were first incubated for 48 hours at 4°C in thegoat antiserum against CTb referred to above, at a 1:5,000dilution, followed by an Alexa 488 donkey anti-goat anti-body (Molecular Probes, Carlsbad, CA), at a 1:200 dilu-tion, for 1 hour. After repeated washes, one in every threesections was incubated in an anti-MOR antibody, at a1:5,000 dilution, or in an anti-NK1 receptor antibody, at a1:3,000 dilution, both raised in rabbit, or in an anti-GABAB receptor, at a 1:5,000 dilution, raised in guineapig. All incubations lasted for 48 hours at 4°C.

The sections incubated in anti-MOR or anti-NK1 werethen immersed in a biotinylated swine anti-rabbit anti-serum (Vector), whereas the sections incubated with theanti-GABAB antiserum were immersed in biotinylateddonkey anti-guinea pig antiserum (Chemicon). All the sec-ondary antibodies were used at a 1:200 dilution and incu-bations lasted for 1 hour. The sections were then incu-bated with Alexa streptavidin 594 (Molecular Probes), at a1:1,000 dilution, for 1 hour, washed, mounted on gelatin-coated slides, coverslipped with buffered glycerol, and ob-served over the subsequent 48 hours.

For each double-immunostaining procedure (CTb plusMOR, CTb plus GABAB or CTb plus NK1), excitationwavelengths of 450–490 nm or 534–558 nm were used toobserve CTb or receptor immunoreactions, respectively.Neurons single-stained for CTb or for each receptor ordouble-stained for CTb and MOR, CTb and GABAB, orCTb and NK1 were counted in the following areas: 1)RVM, including the nucleus raphe magnus, nucleus para-

gigantocellularis lateralis, and nucleus gigantocellularis,pars �; 2) DRt; 3) Sol; 4) VRt; and 5) VLMlat (reticularformation lying between the lateral reticular nucleus andthe caudal part of the spinal trigeminal nucleus). In eachregion, the percentages of neurons double-labeled for CTband each receptor were calculated both in terms of thetotal of neurons immunoreactive (IR) for each receptorand in terms of the total of CTb-labeled neurons. Imageacquisition was performed with a Bio-Rad (Hercules, CA)MRC 1024 confocal system connected to the fluorescencemicroscope referred to above with a single optical sectionused for documentation purposes.

GABAergic control of MOR-expressingDRt neurons

Two studies were performed: 1) analysis of co-expression of MOR and GABAB receptors in DRt neurons,and 2) evaluation of the GABAergic input over DRt neu-rons co-expressing MOR and GABAB receptors. Two addi-tional animals were used in the studies. Following perfu-sion and sectioning as in the double-immunostainingprotocol, one in every fourth medullary section was immu-nocytochemically processed to study the co-expression ofMOR and GABAB receptors in DRt neurons by using animmunofluorescence protocol similar to that describedabove. Briefly, sections were first incubated in the anti-MOR antiserum, at a 1:5,000 dilution, for 48 hours at 4°C,followed by an Alexa 488 goat anti-rabbit antibody (Mo-lecular Probes), at a 1:1,000 dilution, for 1 hour. Afterrepeated washes, sections were incubated in the anti-GABAB receptor raised in guinea pig, at a 1:5,000 dilution,for 48 hours at 4°C, followed by biotinylated donkey anti-guinea pig antiserum (Chemicon) at a 1:200 dilution, andAlexa streptavidin 594 (Molecular Probes), at a 1:1,000dilution, for 1 hour each. After washes, sections weremounted on gelatin-coated slides and coverslipped withbuffered glycerol. Sections encompassing the DRt wereanalyzed at the confocal system described above to iden-tify neurons that co-localized MOR and GABAB receptors.

Study of the GABAergic input over DRt neurons ex-pressing MOR was based on the immunodetection of GADcombined with either MOR immunocytochemistry or bothwith MOR and GABAB immunodetections and used thespare medullary sections of the two animals referredabove. In the first case (GAD plus MOR), the ABC/streptavidin method was used. To inhibit endogenous per-oxidase sections and decrease background staining, incu-bation of primary antibodies was preceded by immersionin a 3.3% H2O2 solution, for 30 minutes, and in the PBSTsolution containing 0.1 M glycine and 10% normal horseserum, for 2 hours. Sections were incubated overnight inthe anti-GAD monoclonal antibody, at a 1:100 dilution.Following washes, sections were incubated in biotinylatedhorse anti-mouse antibody at a 1:200 dilution, for 1 hourand processed by the ABC method (Vectastain Elite, Vec-tor) by using DAB as the chromogen, as described above.Following washes, sections were incubated in the anti-MOR antiserum referred to above, at a 1:5,000 dilution,overnight, followed by biotinylated swine anti-rabbit an-tibody (Dako, Glostrup, Denmark) at a 1:200 dilution, for1 hour. The immunoreaction product was revealed by us-ing a streptavidin-alkaline phosphatase reagent (Vector),at a 1:1,000 dilution, for 1 hour, followed by Fast Bluechromogen (Pinto et al., 2003).


178 M. PINTO ET AL.

Sections were mounted on gelatin-coated slides, clearedin Histoclear, and coverslipped with Permount. Sectionsencompassing the DRt were observed by using a lightmicroscope at 100� to search for appositions of GAD-IRfiber varicosities to MOR-IR neurons. According to previ-ous criteria (Miletic et al., 1984), the existence of apposi-tions was considered if the fiber varicosity and theperikarya profile were in the same plane of focus, with nodiscernible space between.

For the immunodetection of GAD combined with MORand GABAB immunoreactions, a triple immunofluores-cence method was used based on the general immunoflu-orescence protocols described above. Briefly, the sectionswere incubated for 48 hours at 4°C in the guinea piganti-GABAB receptor antibody, washed, and incubated inthe rabbit anti-MOR antiserum, both at a 1:5,000 dilution.Following washing, the sections were incubated in themonoclonal antibody anti-GAD, at a 1:100 dilution. Afterwashes, sections were incubated, for 1 hour, with a bio-tinylated donkey anti-guinea pig antibody (Chemicon) at a1:200 dilution, followed by Alexa 647-conjugated goatanti-rabbit (Molecular Probes) and Alexa 594-conjugatedgoat anti-mouse (Molecular Probes) antibodies, both at a1:1,00 dilution. Then the sections were incubated for 1hour with Alexa streptavidin 488 (Molecular Probes), at a1:1,000 dilution, washed, mounted on gelatin-coatedslides, and coverslipped with buffered glycerol.

Sections were analyzed as described above to search forappositions of GAD-IR fiber varicosities to DRt neuronsdouble-immunostained for MOR- and GABAB-IR. Thesoftware Adobe Photoshop vCS3 (San Jose, CA) was usedfor minor adjustments of brightness and contrast for thesake of equalizing each panel of figures and for addinglabels.

RESULTS

Injection sites and CTb-labeled neurons atthe medulla oblongata

The injection consisted of a homogeneous, compact coresurrounded by a halo in which dark areas intermingledwith lighter zones (Fig. 2A,B). More peripherally, a fewcells were labeled, probably indicating uptake from thecore. Only the central and peripheral areas were consid-ered the injection site (Tavares and Lima, 1994). Theinjections extended from the rostral limit of the C4 seg-ment to the caudal level of the C7 segment. At the spinalgray, the injection sites were restricted to the dorsal hornand encompassed laminae I–V. The injection sites occa-sionally extended to the overlying dorsal funiculus, prob-ably due to tracer backflow, but this does not induce ret-rograde labeling (Tavares and Lima, 1994).

CTb-labeled cells occurred in all the medullary areasunder analysis (Fig. 2C,D) and were recognized by thegreen fluorescence of perikarya and proximal dendrites. Atotal of 9,897 CTb-labeled neurons were counted, largelyprevailing at the RVM (about 76% of the labeled neurons),followed by the DRt (20%), Sol (3%), VRt (3.5%), andVLMlat (0.5%).

Expression of MOR, GABAB, or NK1

Neurons IR for MOR (Figs. 3A,B, 4B), GABAB (Figs.3C,D, 4E), or NK1 (Figs. 3E,F, 4H) were identified by thered fluorescence of the perikarya and proximal dendrites,

which extended to second-order dendrites in the case ofNK1 receptors.

MOR. A total of 9,102 MOR-IR neurons were counted.Their distribution at the pertinent medullary areas isshown in Figure 3A and B and generally agrees with theresults of previous studies (Arvidsson et al., 1995), with aprevalence at the RVM.

GABAB receptors. A total of 15,148 GABAB-IR neu-rons were counted at the medullary areas explored here,with the distribution depicted in Figure 3C and D. Aprevalence of GABAB-IR neurons at the RVM was de-tected, in agreement with results of previous studies(Margeta-Mitrovic et al., 1999).

NK1 receptors. A total of 6,283 NK1-IR neurons werecounted in the medulla oblongata, according to the distri-bution shown in Figure 3E and F. The large prevalence ofNK1-IR neurons detected at the RVM was not referred topreviously (Nakaya et al., 1994), because the medullaryregions analyzed here were not compared in detail.

Double-labeling for CTb and MOR, GABAB,or NK1

Double-immunostained neurons for CTb and each re-ceptor were recognized by the yellowish fluorescence ofperikarya and dendrites. After conversion of the images tomagenta and green, the double-immunostained neuronsappear white (Fig. 4C,F,I).

For all the receptors, most of the double-labeled neuronsoccurred at the RVM (76%) followed by the DRt (16%). Thetotal numbers of double-labeled neurons for CTb and eachreceptor are shown in Tables 1 and 2. The proportions ofneurons expressing MOR, GABAB, or NK1 and projectingto the spinal cord are shown in Table 1 and analyzedbelow. The percentages of spinally projecting neurons thatexpressed each receptor are depicted in Table 2 and ana-lyzed below.

Projection to the spinal dorsal horn of MOR-,

GABAB-, or NK1-expressing neurons

MOR. The VRt, RVM, and DRt were the areas withhigher percentages of MOR-expressing cells that were alsoCTb-labeled, followed by the VLMlat and Sol.

GABAB receptors. The VLMlat, DRt, and RVM werethe areas with higher percentages of GABAB-expressingcells that were also CTb-labeled. Only a few spinally pro-jecting GABAB-IR cells were detected at the Sol and VRt.

NK1 receptors. The VRt and DRt were the medullaryareas with higher proportions of NK1-expressing neuronsthat projected to the cord, followed by the Sol, RVM, andVLMlat.

Expression of MOR, GABAB, or NK1 in spinally pro-

jecting neurons

MOR. The percentages of CTb-labeled neurons thatexhibited MOR were very high at the DRt, RVM, and VRtand moderate at the Sol and VLMlat.

GABAB receptors. The percentages of CTb-labeledneurons that expressed GABAB receptors were over 90%in all areas addressed.

NK1 receptors. The percentages of CTb-labeled neu-rons that expressed NK1 receptors were also high at theDRt, VRt, and RVM and moderate at the Sol and VLMlat.

GABAergic control of MOR-expressingDRt neurons

In sections used to search for co-expression of MOR andGABAB in DRt neurons, MOR-IR neurons were recognized



Fig. 2. A,B: CTb injection site at the spinal cord. The injection siteincludes a central core (in black in A; delimited by white dashes in B)and a peripheral zone (in gray in A) in which dark and light zonesintermingle. C,D: Schematics depicting the distribution of retro-gradely labeled neurons at two rostrocaudal levels of the medullaoblongata of a representative animal. The distance in millimetersfrom the interaural line is indicated at the left, according to Paxinos

and Watson (1998). The areas analyzed in this study are delimited bylarge dashes and include the rostroventromedial medulla (RVM),dorsal reticular nucleus (DRt), nucleus of the solitary tract (Sol),ventral reticular nucleus (VRt), and lateralmost portion of the caudalventrolateral medulla (VLMlat). 1 dot, 5 CTb-labeled neurons; 1 dia-mond, 50 CTb-labeled cells. Scale bar � 100 �m in B (applies to A,B).

by green fluorescence (Fig. 5A) whereas those IR forGABAB were identified by red fluorescence (Fig. 5B). Neu-rons IR for both receptors were very common at the DRt(Fig. 5C). The existence of a strong GABAergic controlover MOR-expressing DRt neurons was further confirmedby the demonstration of GAD-IR fibers upon MOR-IR neu-rons (Fig. 5D) and neurons that co-expressed MOR andGABAB receptors (Fig. 5E).

DISCUSSION

Technical considerations

The present study combined retrograde tracing withCTb with immunodetection of MOR, GABAB, or NK1 re-ceptors. CTb was elected because it allows intense retro-grade labeling with moderate interindividual variations(Almeida et al., 1993; Tavares and Lima, 1994). This wasconfirmed in the present study because the absolute num-bers of CTb-labeled neurons in the different animals weresimilar for each type of double immunoreaction if the finalnumbers of sections counted were considered (Table 2). Ifthis issue is considered, the numbers of CTb-labeled neu-

rons were also similar between the three double-immunolabeling procedures, discarding major interfer-ences between the two immunoreactions (CTb plusreceptors).

As usual in tract-tracing studies dealing with receptorexpression (Kalyuzhny et al., 1996, 1998; Wang and Wes-sendorf, 1999; Yang et al., 2002), the data are not amena-ble to statistical analysis but show regional differences inthe participation of MOR, GABAB, and NK1 receptors inthe circuits involved in descending modulation. However,a detailed analysis of the absolute numbers of neuronsand its relation with the final number of sections used forcounting reinforces the rationale of using percentage anal-ysis of the data in studies dealing with receptor expressionin retrogradely labeled neurons.

General findings and topographic analysis

This study demonstrates variable degrees of expressionof MOR, GABAB, and NK1 receptors in spinally projectingneurons of major medullary pain control centers. Becausehigh proportions of receptor-expressing neurons were notretrogradely labeled, especially in the case of GABAB re-

Fig. 3. Schematics depicting the distribution of neurons IR for MOR (A,B), GABAB (C,D), and NK1(E,F) receptors in the areas delimited in Figure 2C and D. 1 dot, 5 labeled neurons; 1 triangle, 20 labeledcells; 1 star, 100 labeled neurons.



ceptors, it is possible that the receptors are also expressedby interneurons that indirectly control descending modu-lation. Very high proportions of spinally projecting neu-rons expressed the receptors in all the pain control cen-ters, indicating co-expression of the receptors indescending pathways. This was here demonstrated by theco-localization of MOR and GABAB receptors at the DRt.Besides confirming that descending pain control isstrongly modulated by opioids and GABA (Heinricher etal., 1991, 2003; Kalyuzhny and Wessendorf, 1997; re-viewed by Millan, 2002; Gebhart, 2004), the data suggestthat SP also plays an important role in descending mod-ulation because the proportions of NK1-expressing neu-rons retrogradely labeled from the spinal cord were, ingeneral, higher than those obtained for MOR or GABABreceptors.

This is interesting in light of the participation of NK1receptors in central sensitization, which is prevented bydestruction of NK1-expressing spinal neurons, probablydue to disruption of descending effects of spinobulbar

loops (Manthy et al., 1997; Nichols et al., 1999; Suzuki etal., 2002; Khasabov et al., 2002, 2005; Castro et al.,2006b). NK1 receptors are expressed in the ascendingbranches connecting the DRt, the Sol, and the VLMlatwith the spinal dorsal horn (Tavares and Lima, 1994,2002; Todd et al., 2000; Gamboa-Esteves et al., 2001, 2004;reviewed by Lima and Almeida, 2002; Castro et al.,2006a). The present study shows, for the first time, thatNK1 receptors are also expressed in the descendingbranches of the loops that connect the DRt, Sol, and VLM-lat with the spinal dorsal horn.

The present study supports a major role of the RVM indescending pain modulation because this was the medul-lary area containing the majority of labeled neurons. Animportant participation of the DRt is also indicated be-cause it was the second area with (by far) higher numbersof spinally projecting neurons that expressed the recep-tors. At the RVM, DRt, and VRt about 42% of MOR-expressing neurons were retrogradely labeled, whereasabout 92% of spinally projecting neurons expressed MOR.

Fig. 4. Photomicrographs of medullary sections double-immunoreacted for CTb (A,D,G) and MOR(B), GABAB (E), and NK1 (H). C,F,I: Neurons double-labeled (arrows) for CTb and MOR at the RVM (C),CTb and GABAB at the DRt (F), and CTb and NK1 at the VLMlat (I). For abbreviations, see list. Scalebar � 50 �m in C (applies to A–C), F (applies to D–F), and I (applies to G–I).


182 M. PINTO ET AL.

It is likely that in those pain control areas MOR neuronsparticipate directly and indirectly in descending modula-tion, as demonstrated for the RVM in anatomic (Kaly-uzhny et al., 1996) and electrophysiologic (Finnegan et al.,2004) studies and recently suggested for the DRt in apharmacological analysis (Pinto et al., 2006). Similaranalysis of the Sol and VLMlat showed that only 20% ofMOR-expressing neurons were labeled from the spinalcord, favoring a mainly indirect participation of thesereceptors in descending pain modulation. These possi-bilities need to be ascertained by detailed functionalstudies.

Role of MOR, GABAB, and NK1 in paincontrol actions of the RVM

The RVM is the only medullary pain control regionpreviously studied in terms of the expression of receptorsin descending pathways, but only for MOR (Kalyuzhny etal., 1996, 1998; Wang and Wessendorf, 1999; Marinelli etal., 2002; Yang et al., 2002). These studies correlated thedual role of the RVM in pain modulation with MOR ex-pression in spinally projecting neurons (reviewed by Hein-richer et al., 2003). The ON-cells, which were proposed toparticipate in descending pain facilitation, are directly

TABLE 1. Percentages of Neurons Expressing MOR, GABAB Receptor, or NK1 Receptor That Project to the Spinal Cord From the RVM,DRt, Sol, VRt, and VLMlat

Medullary areaExp. no.[sections]

% Double-labeled/MOR

Exp. no.[sections]

% Double-labeled/GABAB

Exp. no.[sections]

% Double-labeled/NK1

RVM 2 [18] 17.7 (294/1,664) 1 [21] 2.6 (51/1.984) 1 [11] 45.5 (245/539)5 [15] 20.1 (345/1,714) 2 [19] 44.5 (793/1.784) 2 [15] 31.9 (208/652)7 [16] 37.6 (692/1,160) 3 [19] 36.2 (1053/2.905) 3 [22] 45.9 (929/2,025)8 [18] 53.2 (973/1,829) 4 [14] 22.1 (383/1.733) 4 [19] 41.4 (670/1,617)

5 [26] 12.2 (345/2.820)Total 36.1% (2,304/6,367) 23.4% (2,625/11,226) 42.5% (2,052/4,833)DRt 2 [1] 24.3 (18/74) 1[5] 0.6 (2/316) 1[1] 0 (0/30)

5 [3] 14.9 (69/472) 2 [5] 42.3 (105/248) 2 [2] 64.3 (36/56)7 [4] 32.3 (203/629) 3 [5] 24.1 (160/663) 3 [4] 74.9 (191/255)8 [4] 43.7 (256/586) 4 [5] 33.9 (236/657) 4 [6] 46.4 (234/504)

Total 31.0% (546/1,761) 26.7% (503/1,884) 53.4% (451/845)Sol 2 [1] 41.0 (16/39) 1[5] 0 (0/273) 1[1] 0 (0/0)

5 [3] 1.6 (5/315) 2 [5] 8.2 (27/328) 2 [2] 47.4 (9/19)7 [4] 1.7 (4/236) 3 [5] 2.7 (15/560) 3 [4] 65.5 (74/113)8 [4] 18.1 (28/155) 4 [5] 5.0 (22/438) 4 [6] 36.4 (32/88)

Total 7.1% (53/745) 4.0% (64/1,599) 52.3% (115/220)VRt 2 [1] 0 (0/0) 1[5] 0 (0/164) 1[1] 0 (0/0)

5 [3] 10.3 (12/117) 2 [5] 3.4 (4/117) 2 [2] 67.3 (37/55)7 [4] 28.6 (10/35) 3 [5] 2.1 (2/94) 3 [4] 57.9 (121/219)8 [4] 70 (42/60) 4 [5] 30.8 (8/26) 4 [6] 80 (56/70)

Total 30.2% (64/212) 3.5% (14/401) 62.2% (214/344)VLMlat 2 [1] 100 (2/2) 1[5] 0 (4/0) 1[1] 0 (0/0)

5 [3] 12.5 (1/8) 2 [5] 100 (1/1) 2 [2] 60.0 (3/5)7 [4] 14.3 (1/7) 3 [5] 60 (3/5) 3 [4] 23.5 (4/19)8 [4] 0 (0/0) 4 [5] 21.9 (7/32) 4 [6] 41.2 (7/17)

Total 22.2% (4/18) 39.5% (15/38) 34.1% (14/41)

Between brackets are the numbers of sections used in each experiment. Between parentheses are the numbers of cells double-labeled for CTb and each receptor and the totalnumber of neurons labeled for the respective receptor. For abbreviations, see list.

TABLE 2. Percentages of CTb-Labeled Neurons Expressing MOR, GABAB Receptor, or NK1 Receptor in the RVM, DRt, Sol, VRt, and VLMlat

Medullaryarea

Exp. no.[sections]

% Double-labeledMOR/CTb

Exp. no.[sections]

% Double-labeledGABAB/CTb

Exp. no.[sections]

% Double-labeledNK1/CTb

RVM 2 [18] 77.4 (294/380) 1 [21] 100 (51/51) 1 [11] 96.1 (245/255)5 [15] 96.9 (345/356) 2 [19] 94.9 (793/836) 2 [15] 45.5 (208/457)7 [16] 98.0 (692/706) 3 [19] 100 (1053/1,053) 3 [22] 90.5 (929/1,026)8 [18] 100 (973/973) 4 [14] 100 (383/383) 4 [19] 93.7 (670/715)

5 [26] 88.0 (345/392)Total 95.4% (2,304/2,415) 96.7% (2,625/2,715) 83.7% (2,052/2,453)DRt 2 [1] 85.7 (18/21) 1[5] 100 (2/2) 1[1] 0 (0/0)

5 [3] 97.2 (69/71) 2 [5] 98.1 (105/107) 2 [2] 51.4 (36/70)7 [4] 100 (203/203) 3 [5] 100 (160/160) 3 [4] 87.2 (191/219)8 [4] 100 (256/256) 4 [5] 100 (236/236) 4 [6] 97.5 (234/240)

Total 99% (546/551) 99.6% (503/505) 87.1% (461/529)Sol 2 [1] 84.2 (16/19) 1[5] 0 (0/0) 1[1] 0 (0/0)

5 [3] 71.4 (5/7) 2 [5] 84.4 (27/32) 2 [2] 26.5 (9/34)7 [4] 100 (4/4) 3 [5] 100 (15/15) 3 [4] 66.1 (74/112)8 [4] 100 (28/28) 4 [5] 100 (22/22) 4 [6] 100 (32/32)

Total 91.4% (53/58) 92.8% (64/69) 64.6% (115/178)VRt 2 [1] 0 (0/0) 1[5] 0 (0/0) 1[1] 0 (0/0)

5 [3] 100 (12/12) 2 [5] 100 (4/4) 2 [2] 57.8 (37/64)7 [4] 100 (10/10) 3 [5] 100(2/2) 3 [4] 86.4 (121/140)8 [4] 100 (42/42) 4 [5] 100 (8/8) 4 [6] 98.2 (56/57)

Total 100% (64/64) 100% (14/14) 82% (214/261)VLMlat 2 [1] 22.2 (2/9) 1[5] 100 (4/4) 1[1] 0 (0/0)

5 [3] 100 (1/1) 2 [5] 16.7 (1/6) 2 [2] 16.7 (3/18)7 [4] 100 (1/1) 3 [5] 100 (3/3) 3 [4] 44.4 (4/9)8 [4] 0 (0/0) 4 [5] 100 (7/7) 4 [6] 100 (7/7)

Total 36.4% (4/11) 93.7% (15/16) 41.2% (14/34)

Between brackets are the numbers of sections used in each experiment. Between parentheses are the numbers of cells double-labeled for CTb and each receptor and the totalnumbers of CTb-labeled neurons in that area. For abbreviations, see list.



inhibited by opiates, namely, those acting on MOR. TheOFF-cells, which appear to be associated with pain inhi-bition, do not express MOR and are indirectly excited byopiates (Fields et al., 1991; Heinricher et al., 1992, 1994,

1999). The functional relevance of these observations forpain control has been demonstrated by showing that se-lective ablation of MOR-expressing ON-cells inhibits neu-ropathic pain (Porreca et al., 2001). According to the

Fig. 5. GABAergic control of MOR-expressing DRt neurons. A–C:Co-expression of MOR and GABAB receptors in DRt neurons (A,MOR-IR neurons; B, GABAB-IR neurons; C, merged image of A and B;double-labeled neurons are marked with arrows). D,E: GABAergicinput over MOR-expressing DRt neurons (D) or neurons that co-express MOR and GABAB (E). D shows several GAD-IR varicosities

(brown), some of which are in close apposition (arrows) to a MOR-IRneuron (blue). E shows GAD-IR varicosities (red), some of which are inclose apposition (arrows) to DRt neurons double-labeled for MOR(blue) and GABAB (green). For abbreviations, see list. Scale bar � 100�m in C (applies to A–C) and D; 50 �m in E.


184 M. PINTO ET AL.

present results, about half of the MOR-expressing RVMneurons were retrogradely labeled from the cord, probablycorresponding to ON-cells involved in direct pain facilita-tion. However, MOR-expressing RVM neurons were alsoproposed to be interneurons that control spinally project-ing neurons, some of which are OFF-cells (Finnegan et al.,2004). It would be interesting to ascertain whether theMOR-expressing RVM neurons not retrogradely labeledfrom the spinal cord correspond to those interneurons.

In addition to MOR, our results suggest that GABABand NK1 receptors may also control descending modula-tion from the RVM. Interestingly, only 22% of GABAB-expressing neurons in the RVM were retrogradely labeled,which could account for the dual effect of the GABABagonist baclofen with antinociception at low doses andhyperalgesia at high doses (Thomas et al., 1995). A circuitorganization in which RVM interneurons with GABABreceptors inhibit spinally projecting antinociceptive (OFF-cells) neurons would explain the dual effects of baclofen(Thomas et al., 1995) is proposed in Figure 6. As to NK1receptors, it was recently demonstrated that its agonistsincrease the activity of ON-cells but have no effect onOFF-cells, indicating that NK1 receptors are preferen-tially located on ON-cells (Budai et al., 2007). The possi-bility that MOR and NK1 receptors may be co-expressedby ON-cells is supported by our results showing similardegrees of expression of MOR and NK1 receptors in neu-rons of the RVM-spinal pathway. The co-expression of

MOR and NK1 supports previous proposals that NK1receptors are involved in opioidergic modulation. The an-algesia induced by icv injection of NK1 agonists is re-versed by naloxone (Improta and Brocado, 2000; Rosen etal., 2004), and systemic morphine induces the release ofSP at the PAG (Rosen et al., 2004).

The present anatomical results raise several possibili-ties for the participation of MOR, GABAB, and NK1 indescending pain control from the RVM (Fig. 6). Althoughthe existence of of pain-inhibiting (OFF-cells) and pain-facilitatory (ON-cells) neurons in the RVM has never beenchallenged, the proposed circuit does not consider otherfunctions of the RVM besides pain control. Based on re-cent proposals that the RVM controls several homeostaticphysiological processes (Mason, 2005a,b), it is possiblethat the differential expression of MOR, GABAB, and NK1in spinally projecting RVM neurons has repercussions inother functions besides pain control.

Opioidergic and GABAergic inhibition ofdescending facilitation from the DRt

A novel finding of this study is the relevance of the opioi-dergic and GABAergic inhibition of the pronociceptive ac-tions of the DRt. These are known to be conveyed by a closedreciprocal spino-DRt-spinal loop, but the mechanisms thatcontrol this amplification system, already postulated (Limaand Almeida, 2002), remained unknown. As discussedabove, the present results suggest that MOR may directly

Fig. 6. A model of proposed circuits centered at the RVM and theDRt that mediate descending modulation of nociceptive transmissionand include MOR, GABAB, or NK1 receptors. The diagram was con-structed based on the present results and also taking into accountprevious findings (see text for references). The descending facilitatoryeffects (�) exerted by the DRt are proposed to be controlled by opi-

oidergic and GABAergic inhibition (�) of spinally projecting DRtneurons. This balance between opioidergic and GABAergic control ofdescending modulation is also common to the RVM. An importantparticipation of NK1 receptors in descending modulation from theRVM and DRt should be considered. For abbreviations, see list.



and indirectly participate in the descending modulation fromthe DRt. We recently demonstrated that microinjection ofthe MOR agonist DAMGO into the DRt induced hyperalge-sia (Pinto et al., 2006) probably due to inhibition of MOR-expressing inhibitory neurons that act upon the DRt facili-tatory cells. Figure 6 presents a schematic of how opioid andGABAergic circuitry regulates the pronociceptive effects ofDRt activation. It is likely that GABA also plays an impor-tant role in modulating the net descending input from theDRt based on the co-expression of MOR and GABAB recep-tors demonstrated in the present study. It is important toevaluate pharmacologically the interactions of opioids andGABA in the inhibition of descending facilitation from theDRt. This may be relevant for chronic pain control becausethis situation is associated with increase of descending facil-itation (Ossipov and Porreca, 2006), and increased facilita-tion from the DRt was recently shown to occur in chronicpain models (Sotgiu et al., 2008).

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participation of μ-opioid, gabab, and nk1 receptors of major pain control medullary areas in...

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