neuromedin-N mRNA expression: a hybridization-histochemical and immunohistochemical study using three different rat models for chronic nociception

Brain Research, 611 (1993) 87-102 Cl1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

87

BRES 18795

Chronic pain increases brainstem proneurotensinjneuromedin-N mRNA expression: a hybridization-histochemical and immunohistochemical study using three 'different rat models for chronic nociception Frank G. Williams and Alvin J. Beitz Department of Veterinary Biology, College of Veterinary Medicine, University of Minnesota, MN 55108 (USA) (Accepted 8 December 1992)

Key words: Inflammation; Mononeuropathy; Hyperalgesia; Arthritis; Neurotensin; Periaqueductal gray; Nucleus cuneiformis

!~e role of neurotensin in the central nervous system is poorly understood. Exogenous neurotensin has potent anti nociceptive effects when Injected into the midbrain periaqueductal gray (PAG). Although it is present in terminals, fibers and perikarya within the PAG and other Illidbrain regions known for their antinociceptive circuits, it is not known whether endogenous neurotensin modulates nociception. We examined the midbrain in three different rat models for nociception to learn whether acute or chronic pain altered neuronal levels of the ~roneurotensin/neuromedin N mRNA (neurotensin mRNA). The models were: adjuvant-induced polyarthritis, adjuvant-induced unilateral paw Inflammation, and unilateral peripheral mononeuropathy caused by ligation of the sciatic nerve. Behavioral observations confirmed that the e~pected symptoms developed as previously described. Within each of the three experimental models, we performed in situ hybridization histochemistry on coronal sections from three midbrain levels that included the rostral one-third of the PAG, the middle one-third of the PAG, and the caudal one-third of the PAG. At the level of the rostral PAG, we found that neither chronic nor acute nociception altered the frequencies or distributions of neurons containing neurotensin mRNA. In contrast, at the levels of the mid- and caudal one third of the PAG, the early effects of the nociceptive lesions differed from the chronic effects. During the acute phase of each model, increases in either the frequency or field area of neurons that were hybridization-positive for neurotensin mRNA were confined to the ventromedial PAG and the dorsal raphe nUcleus. As the nociceptive stimuli became chronic, the early increases in neurotensin mRNA-containing neurons at the level of the middle third of the ventral PAG were diminished but remained above control levels, while increases in neurotensin m.RNA began to occur in the midbrain tegmentum lateral to the PAG. The most striking increases in neurotensin mRNA expression were observed 16-17 days after the onset of nociceptive stimuli. At the level of the mid-PAG and caudal PAG, increased hybridization signal intensities and neuron frequencies occurred Within the nucleus cuneiformis and the lateral tegmental nuclei, including the pedunculopontine and microcellular tegmental nuclei, as well as the deep mesencephalic nuclei. Hybridization-positive neurons in the tegmental nuclei were not observed at early stages of lesion development, but were a consistent feature of caudal midbrains after nociception became chronic. The finding that early and late effects of nociception were Clearly different suggests that a unique subpopulation of midbrain neurons adapts to pain by producing mRNA that codes for a putative ~ntinociceptive peptide. To confirm that increased neurotensin levels accompanied the increased mRNA levels, we performed immunocytochemIstry on coronal sections from the peripheral mononeuropathy experimental group and quantitated the immunoreactivity of fibers and terminals USing an image analyzer. As expected, statistically significant increases in peptide levels were observed in the PAG and nucleus cuneiformis. These studies suggest that chronic nociception can produce changes in the distributions and cellular levels of an antinociceptive peptide. We COnclude that neurotensin in the caudal midbrain may modulate nociception, and that different areas of the midbrain may modulate acute vs. chronic nociception. '

INTRODUCTION

Since descending pain modulation systems were first reported by Reynolds 41 , it has been hoped that these endogenous systems could be manipulated to produce Clinically useful antinociception. This goal has engendered numerous investigations into the anatomical or-

-

ganization and neurochemical properties of the anti nociceptive pathways (recently reviewed in refs. 9 and 23). Among the constituents of these systems, the periaqueductal gray matter (PAG) is uniquely situated to play an important role in descending antinociception 4 ,47. The PAG receives input via both spinal and telencephalic projections and sends efferents to ventral

Correspondence: F.G. Williams, Department of Veterinary Biology, College of Veterinary Medicine, University of Minnesota, 295 AS/VM, 1988 Fitch Avenue, St. Paul, MN 55108, USA. Fax: (1) (612) 625 0204.

88 medullary regions that are critical to descending antinociceptive activity. In addition to the PAG, several 4lther regions of the midbrain have been scrutinized because of their anatomical relationship to the PAG or to the ventromedial medulla. The nucleus cuneiformis, for example, lies lateral to the PAG at the level of the caudal midbrain. Stimulation of the nucleus cuneiformis results in a' profound increase in nociceptive tailflick latencies 47 and inhibits the response of dorsal horn nociceptive neurons to noxious stimulation 14.24. Previous studies have shown that this region, like the PAG, has a substantial projection to the ventromedial medulla 7,47 and that it produces both mono- and polysynaptic effects on neurons in the ventromedial medulla 47• The tridecapeptide neurotensin is present within terminals, fibers, and perikarya that are heterogeneously distributed within the caudal midbrain 29,43,46. The PAG contains the greatest number of neurotensinimmunoreactive structures, which are more abundant within the caudal and ventral portions of this region. Several lines of evidence suggest that neurotensin might modulate nociception. It can produce a long-lasting ·· d 3038 analgesia when central Iy a d mmlstere ' . Neurotensin injected into the PAG caused potent analgesia, excited PAG neurons, and increased the firing rate of neurons in the rostroventral medulla l ,5,6. This is significant because spinal projections from the rostroventral medulla are thought to mediate antinociception. Further, binding sites for radiolabeled neurotensin in the caudal midbrain are consistent with observed locations of terminal fields; the PAG, the substantia nigra, and midline nuclei ventral to the aqueduct all bind radiolabeled neurotensin 37• Despite the potent antinociceptive actions of exogenous neurotensin, the role of the endogenous peptide within the central nervous system remains to be demonstrated. Thus, the general goal of these studies was to learn whether chronic or acute nociception can affect midbrain neurons that synthesize neurotensin. Specifically, we wanted to know whether acute vs. chronic nociception altered neurotensin biosynthesis and storage within the midbrain, and whether the effects occurred in regions known to be involved in antinociception. To address this question we performed in situ hybridizations to examine the production of proneurotensin/neuromedin N mRNA (neurotensin mRNA) in the midbrain. In addition, we confirmed that the changes in mRNA production were associated with alterations in immunoreactive neurotensin peptide levels. Our hybridization studies were performed using three different models for chronic nociception because

complications due to stress or physiologic responses to nociceptive lesions are difficult to control. Polyarthritis, secondary to inflammation caused by adjuvant inoculation, has been well documented to be associated with chronic nociception17. Long-lasting hyperalgesia and other behavioral manifestations of nociception develop at different intervals, beginning earlier than 7 days following inocuhition and peaking approximately 21 days after inoculation. Unilateral inflammation of one hind paw has been proposed as an alternative to .. 2844 poIyart hntis ' ,an d'IS a dvantageous because the nOCI-. ceptive lesion is localized to a single peripheral site, rather than affecting both hind limbs, tail, and pelvis. Intraplantar inoculation with adjuvant causes increased paw volumes and hyperalgesia beginning several hours after inoculation. The hyperalgesia reportedly lasts beyond one month, permitting studies of long-term nociceptive stimulation. The third model of chronic nociception we studied, experimental peripheral neuropathies, can be produced by chronic constriction of the sciatic nerve to (reviewed in ref. 11). These lesions produce long-lasting symptoms consistent with chronic pain: the paw of the affected limb is guarded soon after recovery from surgery; chronic hyperalgesia develops 1-2 days after surgery and is maintained for approximately 21 days; and spontaneous discharge of small and large myelinated fibers originate in the dorsal root ganglion 1-3 days after surgery. MATERIALS AND METHODS Animal models for chronic nociception Male Sprague-Dawley rats (200-250 g)' were obtained from Sasco Inc. (Omaha, NE). Male Wistar rats (200-225 g) were obtained from Harlan Sprague-Dawley (Indianapolis, IN). All animals were housed 3-4 per cage at 22-24°C, 12 h light/dark cycle, with food and water available ad libitum. Three models for chronic nociception were employed in our studies. Polyarthritis was induced by intradermal injection of Freund's Complete Adjuvant (0.5 mg/ml heat killed Mycobacterium butyricum suspended in 85% oil/15% emulsifier - Difco Detroit MI) at the . 1736 " base of the tall . . To attenuate the loss of weight and other undesirable side effects associated with onset of arthritic pain, Sprague-Dawley animals were compared to Wi star animals in the polyarthritic model. Sprague-Dawley animals received 150 Jotl, Wistar animals received 60 ~I, control animals received an equal volume of mineral oil. Generalized inflammation and retardation of weight gain were reduced in Sprague-Dawley animals 17• Although some effects were observed as early as four days post-inoculation, we never observed hyperventilation or increased water intake until the sixth day following inoculation. Therefore, the seventh day after inoculation was chosen to represent a short-term exposure to polyarthritic pain. Unilateral injection of Freund's adjuvant into the hind paw has been proposed by Stein et al. 44 as an alternative to the polyarthritic model. Male Sprague-Dawley rats received a 100 Jot I intraplantar injection of complete Freund's adjuvant into the right hind paw. Rather than producing T-cell mediated delayed hypersensitivity and bilateral inflammation at mUltiple joints, the injection causes a unilateral lesion that produces hypersensitivity to noxious pressure 44 and heat.

89 Peripheral mononeuropathies produced by loosely constricting the sciatic nerve cause nocifencive behavior and hyperalgesia to noxious radiant heat lO . Male Sprague-Dawley rats (200-250 g b.wt.) Were anesthetized with ketamine/xylazine (100 mg ketamine and 20 mg XYlazine per kg b.wt.), and their sciatic nerves exposed at the mid-thigh. Four loose ligatures of 4-0 chromic gut were placed ?round the sciatic nerve as previously describedlO, spaced at 1.5 mm Intervals, and the incision closed. The presence of hyperalgesia was confirmed by comparing withdrawal latencies to noxious radiant heat b~tween the paws ipsilateral and contralateral to the lesion. Normal ~Ithdrawallatencies ranged from 8-15 s. Our cutoff time to prevent IIssue damage was 25 s. The data were represented as difference SCores: the paw withdrawal latency on the ligated side minus the paw Withdrawal latency on the non-ligated side. Oligonucleotide probes for hybridization histochemistry Several synthetic DNA oligonucleotides were employed for the in situ hybridizations. Rat proneurotensin mRNA was detected using a 45 base synthetic oligonucleotide complementary to bases 708-752 of the rat sequence 31 ; this probe will be referred to as the 'antisense NT' probe. Control hybridizations were performed using a thirty-six base synthetic oligonucleotide matching the sequence of canine enteric neurotensin cDNA bases 491-526 19• Both oligonucleotides Were synthesized from !3-cyanoethylphosphoramidites, purified by HPLC, and end-labeled using terminal transferase (Boehringer Mannheim, Indianapolis, IN) and biotinylated dUTP (bio-16-dUTP, Boehringer Mannheim, Indianapolis, IN). Biotinylation reactions Were verified by removing 1 fJ-l aliquots of the reaction mixture b.efore, during, and after the terminal transferase reaction and blotling them onto dry nitrocellulose. Biotin on the nitrocellulose membrane was detected using alkaline phosphatase and reagents and procedures from Vector Laboratories (Burlingame, CA). Melting Points for the probes were calculated for 4 X SSC and 50% formamide according to an empirical formula 2 • The melting points Were: 51.soC for the 45 base antisense NT probe and 41.5°C for the 36-base NT control probe. In situ Hybridization methods The hybridization methods employed in these studies were similar to those described previously 45. Briefly, animals were sacrificed by decapitation and brains were rapidly removed and frozen using freon and dry ice. Cryosections (15 fJ-m thick) were stored up to three days at - 20°C until they were processed for hybridization. The Unfixed sections were immersed for 10 min in 4% paraformaldehyde/PBS and rinsed twice for 5 min in PBS. Sections from perfuSion-fixed brains were rinsed twice for 5 min in PBS. All sections were acetylated for 10 min in 0.25% acetic anhydride in 0.1 M triethanolamine hydrochloride/0.9% NaCl. The tissues were then dehydrated and delipidated through graded ethanols (70% to 100%) to chloroform, returned to 95% ethanol, briefly dried, and then stored at - 20°C until used. When the hybridization histochemistry Was performed, the sections were rehydrated with 25 fJ-l of the hybridization mixture lacking only the oligonucleotide probe. That Solution contained 4 X SSC (I X ssc = 150 mM NaCl, 15 mM NaCitrate, pH 7.2), 50% deionized formamide, 10% de!'tran sulfate, 1 X Denhardt's solution (0.02% each of polyvinylpyrrolidone, alkylated and dialyzed bovine serum albumin, and fico))), 0.025% yeast tRNA, 0.05% denatured salmon testis DNA, and 1 mM RNA-vanadyl complexes, and 3 ng/ fJ-l oligodeoxythymidine 12mers. The Oligonucleotide probes were dissolved in hybridization solution at a concentration of 2.5 ng/fJ-l, 5 to 6 ng of probe was added to the solution covering the tissues and each section was covered with parafilm. The slides were incubated in a humidified chamber at 37°C (or 27°C for the NT control probe) for 4 h. The tissues were rinsed twice for 5 min each at 37°C (or 27°C for the NT control probe) in 4 X SSC/50% formamide and once for 15 min at room temperature in 2 X SSC. FITC-avidin DN (Vector Labs, Burlingame, CA) diluted 50:1 in 100 mM bicarbonate buffer, pH 8.5, containing 1% alkylated BSA was applied to the tissue, covered with parafilm, and incubated for 1 h at room temperature in a humidified chamber. Excess FITC-avidin was removed by three 10 min washes in 2 X SSC at room

temperature and the sections were coverslipped in 75% glycerolj25% sodium bicarbonate containing 1% n-propyl gallate 33 for mapping of the fluorescent neurons. . Controls for in situ hybridization In addition to demonstrating that neurotensin peptide and mRNA were co-localized using our immunohistochemical and in situ hybridization techniques 45, three different controls were employed to rule out non-specific interactions between brain sections and the labeled oligonucleotides. First, the 36 base NT control oligonucleotide, having a sequence similar to the peptide coding region of the proneurotensin mRNA, was used according to the above protocol. Second, the thermal stability of the hybrids formed by the 45 base antisense NT oligonucleotide was tested by adding a 30 minute stringency wash in 2 X SSC at 75°C immediately before the FITCavidin incubation. Third, a pre-hybridization digestion with RNase was performed as follows: after immersion fixation of cryosections in 4% paraformaldehyde and PBS rinsing, the tissues were treated with 1 fJ-g/ml proteinase K (Boehringer Mannheim) for 30 min at 37°C, The sections were then re-fixed for 10 min in 4% paraformaldehyde, rinsed twice for 5 min in PBS and covered with hybridization solution (minus RNA-vanadate complexes) containing the biotinylated oligonucleotide plus 20 fJ-g/ml RNase A (Boehringer Mannheim). The remaining steps in the hybridization were performed as described above. Analysis and mapping of hybridization-positive neurons Fluorescent hybrids in tissue sections were viewed on a Nikon Labophot-2 epifluorescence microscope. Mapping was performed by scanning the sections and counting the number of visible hybridization-positive neurons within a defined area on an ocular reticule. The frequency of hybridization-positive cells per area was represented on brain atlas drawings as one of four gray densities. Level one (light gray) equals 1-5 cells/2 X 10 5 fJ-2, level two equals 6-10 cells/2X 10 5 fJ-2, level three equals 11-20 cells/2x 10 5 fJ-2, and level four (the darkest gray) equals > 20 cells/2x 10 5 fJ-2. These four neuron frequencies were drawn into maps using a Macintosh computer running Cricket Draw and Adobe Illustrator (Adobe Systems Inc., Mountain View, CAl software. The purpose of the maps was to summarize a large volume of anatomical information in a semiquantitative format. Based on the map data, certain midbrain regions were chosen for analysis of the intensity of the hybridization signal, rather than the frequency of neurons showing a detectable hybridization signal. These methods employed an image analysis system and are described below. Video microscopy and image analysis Video images were acquired using a Hamamatsu C2400 video camera (Hamamatsu Photonics KK, Hamamatsu City, Japan) and digitized using a Matrox MVP-AT image analysis board (Matrox Electronic Systems, Ltd., Dorval, Quebec, Canada) in a Northgate 386-33 AT-compatible computer (Northgate Computer Systems, Eden Prairie, MN). Images in TIFF format were transferred to a Macintosh via ethernet using Telnet 2.3 and Hyper-FTP software (National Center for Supercomputer Applications, University of Illinois, Urbana, IL; Cornell University, Ithica, NY). Using the Macintosh, image contrast and brightness were adjusted using Photos hop 2.0.1 software (Adobe Systems, Inc., Mountain View, CA) and photographic negatives were made using a Montage FR1 digital film recorder (Presentation Technologies, Sunnyvale, CA) and Kodak T-Max 100 film (Eastman Kodak, Rochester, NY). Image analysis was performed using Image-l software (Universal Imaging Corp., West Chester, PAl. Relative fluorescence intensities were determined by calibrating the response of the video camera and image analysis system using a stable fluorescent source. Neutral density filters, each with known transmission characteristics, attenuated the microscope's emission signal. A calibration curve was obtained by plotting digital image intensity vs. the filter attenuation factor. Computations based on the image data were performed using Microsoft Excel software (Microsoft Corporation., Redmond WA) and statistical analyses were performed using SuperANOVA software (Abacus Concepts, Berkeley, CAl.

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JCllnc:s. Usc of Lhis Bnti body for im munohistochcmiSlrY is described In II .,..evious report from thiS la bonuory .... The In tibody was aoo sucre'isfully llscd to neutrahze: e ndOicnOu$ NT In the ven tral teimenlal nrea. permitllllg an II1Ycslia:nion of ncufOlcnsi n'$ effec ts on dopamineraic neurons Radioimmunoassay data indicate Ihal the a nt ibody reoollnize~ the c:u boxyl te rminU S of th e pe ptide (L Jennes., personal communication)' As described ca rl ic r olll tissue sections we rc: incubated on II rotalin, shaker ove rmght al 4·C wi lh the prnnary an tibody di hu cd I :2.000 In PBS. They were then processed by the mlnlUnopcroxidase·diaminoi>e nl idine
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Animal models We hoped to lessen thc undesirable side effccts that accompany polyarthritis. Encouraging results were ob· tained by simply ch:mgi ng unimal strains. As shown in Fig. lA , weight gain in Sprague- Dawley animals was on ly retarded until the twelfth day following inocu la· lion . Swclling of the hind paw and tarso-tibiu l join t was quan titated by adding Ihe circumference of the two, and is also graphed in Fig. IA. A maximum increase of 0.4 em was observed 8 days after inoculation. This increase is upproxi mately half of th at reported for Wistar anim:ll!. l'. We also observed that hyperventi la· tion, decreases in motility and increases in voca lization we re less seve re in polyartit ritic Sprague- Dawlcy ani· rna Is (dal a no t shown). In an imals receiving unilate ral paw injcclions or sciatic ligatures, hyperalgesia was quantitat ed by COOl puring the lllt ency with which cach hind paw was withdrnwn from noxious radia nt hea l. The paw wit hdrawal resuits, presented in Fig. 113, display a markedly d iffe rent time course between the models. Unilateral puw injection of adjuvant produced a maximal hype ral· gesia 24 h afte r the inoculatio n. At this time point , the IIverage withdrawal latency d iffered by II s between hind paws. The di fference dim inished 10 6 s by the third dflY post-inocula tion, and then declined nea rly lincarly thcrellfter. In contrast to the acute onsct of behavioral symptoms observed afte r paw injections, the wi thdrawal difference scores for animals with scia tic ligations decrcased mo re slowly. Typi c~11 of the data prcsentcd by Bcnnel\ and Xie lO , the maximal behavioral effect was observcd after the fi rst week (Fig. I B). In our animals, the uve rage wit hdrawal difference score lifte r 10 days was - 5.5!1o, li nd thc differences between paws were maint ai ned fo r at least 17 days.

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Fia· I. Time course of behaviorat chanles IlsSOCiated wilh the Ihree models for chronic nocice pho n. A: in poiyarthritic Sprague-Ollwle) rUI S, both the dct rel.ses in weighL 1I1lin (so lid symbols: experimenwlS minus coni rots) lind inneaks in the circumfe rence of Ihe IllfSOlibiRI ,/Olnl and mld·paw (limy symbols; expc: rim enl als minus controls) were maxim.a l eight da)'5 follOWing adjuvant inocula Lion (" _ to). Signifi· ca.nt differences bel\\.'een control and experimenlat aroups., as dclermined by AN?VA Dnd the Newmlll1 - Kculs post. hoc le\l, are indio ca ted by IlSlc nsks. I): lhe lime course wi th which unil ateral lesions affect paw withdr.lw~1 from rudlllu l helll differ belween Imw intlllm' mJ IIOn (solid symbols) and mOnOn eUTOllllthy·bearmg anim als (IIT'JY symbo ls). In bol h of the ullil olerlll models, paw wh hd mwlli latene)' §cores .....ert delemlined ru; the difference betll een Ihe lI\1cmge wl lh· drawal lime of Ihe coni roillaw nlmus the average wlthdrawnl InlcnC)' of the io::sioned p:IW. Significant differences belweel1 ooll irol lind expc:nmenllli ,roups, as dctermmed by ANOVA and the Newmlln~ Kellb post· hoc test, arc indkhled by aSlcrisks.

/-Iybril/iZlIfioll histochemistry

COlllrols, Sever:l! comrols for ncurotc nsi n hybridization have been previously reported 4 \ includ ing illl. munohistochclllistry combined with hybrid ization histo· chcmistry. These siudies confirmed the ce llu lar co-Iocu liZ:llion o f Ile uro tensin im munoreaclivity wi th radio· labeled oligonucleotides in the ra t midbrain . For the present study we pe rformed three negative cont rols, displayed in Fig. 2. These may be compa red 10 Fig. 3A,C,E, which shows typical nuorescent hybridiza tion signa ls at the same magnification. Fig. 2A demonstraled that a ]()()·fold excess of unlabeled oligon u· cleotide eliminated the hybridization of the biotinylated o ligonucleotide. The faint nuorescent signals were not significantly IIbove backgrou nd . The same level of signa l was obse rved using a biotinylntcd oligonu· cleotide Ihat was the inver!toc complement of the :tnli-

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91

sense NT probe (Fig. 28). Pretreatmen t of the sections With ri bon uclease (Fig. 2C) also el iminated or lowered the nuorescent signal. Morphology of h},bridizaliol1·posiliI 1c tlml lIellrolell,~ill­ immunoreactive nel/rOI/S. Video micrographs of neu-

rOtensin neurons detected using hybridiza tion of biOtinylated DNA oligonucleotides or immuno histochcmistry are di splayed in Fig. 3. These exam pl es were taken from Sprague- Dawley ra ts havi ng periphe ral rnononeuropath ics. Neurons in the ventral PAG and dorsal raphe nucleus (Fig. 3A - arrowhea9 on one exa mple) arc small and closely spaced. Immunoreactive neuron perikarya arc equa lly numerous within the same region (Fig. 38 , arrowhead on one exam ple), and immunoreactive terminals (small arrows) and fibers are abu ndant. Within the ven trolat eral PAG, hybridization-positive (Fig. 3C) and neurotensin-immunoreaclive neurons (Fig. 3D) are sma ller and less numerous than in th e ve ntromedial PAG. Neurotensinimmunoreactive fibers and termi nals (small arrow, Fig. 3D) are sligh tly less nume rous as well. Compared to PAG neurotensin hybridiza tion·positive neurons, hybridization-positive ancl immunoreactive neu rOlensi n neurons in the lateral tegmental areas of the midbrai n (Fig. 3E vs. 3A and 3C) are larger and less numerous. Immunoreactive fibers (lnd te rminals arc also abu ndant (Fig. 3F, small arrows). Comparing hybridiza tions to immunohistochemistry, in the three regions shown in Fig. 3 and in ot her areas o f the midbra in there is good cOncorda nce between the morphologies and frequen· eies of hybridizat ion-positive vs. immunoreactive neu· rons. Most immuno reactive structures arc densely· stained fi bers and te rminals, wh ile perikarya sta in ed lightly because no colch icine was used in these experi· Illents. M llf)S

appea red to extend laterally and ventrally into the nucleus euneiformis and tegmental areas. The neuron dist ributions were greatly increased in polyarthritic animals (Fig. 48) com pared to cont rol animals (Fig. 4A). Polya rthritis-induced increases in the densi ty of hybrid ization-positive neurons was most profound in the ventral PAG and dorsal raphe, where the frequency of hybrid ization-posi tive neurons reached its highest level. The spread of hybridizat io n·positive neurons increased rostral to the nucleus cu nciformis and lateral to the

of lIellrolem;in mRNA ill chrOl/ic pain s((j(es

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Hybridization-posi tive neurons, such as Ihose depicted in the micrographs o f Fig. 3, we re mapped to standardized rat brain corona l sectionsJ9 based on their frequency. Four frequency ranges wcre chosen to represe nt the number of hybrid iiation-posilive neurons per area, and clIch range is represented by a different gray halftone densi ty. Maps of typica l neurotensi n mRNA·cont ai ning neuron distribu tions in the cauda l midbrai n of polyart hri tie Wista r rats arc shown in Fig. 4. In cont rol an imals (Fig. 4A) the highest densi ty of neurotensin mR NA-eon taini ng neurons WllS found in the pe riaqued uctal gray and dorsa l raphe. The dorsolateral subdivision of the r AG was spa rsely populated, while the dorsal raphe and ventral PAG contained 11 -20 cellsj2 X lo j Jl z. The group o f hybridiza tion positive neurons in the ventrolateral PAG

lI ~b riui1.ati()n controls show no flilorescence. A: a llJO·fnld of untubctcd oliGonucleotide WIIS audeu to the tissue sectlllil in audition to the normlll amoum of hiotinylateu oligollucleotide. Il: a bioti n~li1 t ed oll~onuelcotidc whru.c sc(,lIcnee mutched the nCIi ' rotcnslII mRNA was u!
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92 PAG , and fr equencies increased in late ral tegmental areas including the pcduncu loponlinc and microcelluJar tegmental nuclei. The frequencies of ncurOleosin mRNA-conlnining

neurons in Wistar anima ls is ge ne rally hi gher than in Sprague- Dawley strain rats. Fig. 5 depicts the (requell-

anatomical region contai ning neurotensin hybridization-positive neurons in control Sprague-Dawley ratS (Fig. SA) also contains hybridization-positive neurons in polyarthrilic animals (Fig. 58 ,C,D). These regions include the PAG , the dorsal raphe nucleus, nucleuS

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cuneiform is, and the latera l tegmenta l regions of the midbrain. A s also seen in the Wistar animals. the

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Fill. 3. Video mIcrographs of nllorescclll hybridi7.lltiuu hislOchemistty :wd immunohistoc hemiwy. Three regions of the ca udal midbrain arc A Iwd U - ve nt ra l t'AG / dorsal mphe nu clo::us: C lind D - ve ntrol:lIeral PAG: E and F - latcral tegmenlftJ region. Fluoresce nce deteclton o r neurolcnsin mRNA in pcri b tya is displayed ~l arrowheads (IHtOels A. C :md E). Im nlUuoh istochemi cal sl:tiui ng for neurotcnsin ( panel~ B. D and FJ revealed both pcrikHrya (arrowheads) Hlld numcwus dcn ~ ly,sI3in ed lerminals (s mall :arrows). SCllie bar ... 25l-' m. d i~pIOl~ed:

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Fig. 4. Maps of represc nta hve hyb ridization-positive ncuron frequen(11 - 3). A; oonlfol animals received injec tions of mlll!;:ral oil ve hicle. B: cKperimcnt al animals we re sacrificed 24 days afler subdennal injection of oom· pletc Fteuml"s AdJu~;lIl t. DR _ dorsal raphe nucleus; r AG .. IlC riaqueductal &ray; SCI' _ ~uperior cerebellar ped uncle; CnF nucle us cuneiform IS; MiT, .. microcellular tegmenta l nucleus.

cies in the e:ludal midbrain of Wistar strain ruts

rons in Sprague- Dawley rill midbrai ns is found in the Ve ntral PAG and do rsa l raphe nucle us. L.·u eral to these regions, hybridiz:tlion-positive neuron frequencies decl ine in the nucleus cuneiformis. NCLLrolc nsin

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hybrid izat ion-positive neurons we re also observed in the region o f the microce llul ar tegme ntal nuclells. In polyarthritic Sprague- Dawley animals, we examined the distributio ns o f hybrid izat ion-positive neu rons at three post-inoculation periods. Each pcriod coincides with our paw and joint in fl ammation and weight gain data (Fig. IA). Four days after inoculation there is no evidence of infl am matory pa in, and changes in hybridization-positive neu ron freq uencies are mino r. Compared to control animals (Fig. 5A). the cuneiform nucle us exhibits a higher ne uro n frequency. while the do rs:1l raphe decreased in neuron frequency (Fig. 5B). Seven days a(ter inocul ation the animals exhibit behavioral evidence o f nocice pt ion. In these animals ( Fig. 5C). cell groups within the late ral PAG and nucleus Cllneiformis have increased in frequency and expanded in area compared to the cont rols (Fig. 5A) and th e ea rl ier post-inoculation period ( Fig. 58 ). More neurons in the late ral tcgmentum O,Hera i to the nucleus cuneiformis) have also become hybridization-positive. This trend cOOl inued 24 days afte r adjuvant inocul ation. T he lateral tegmenlal ne uron group ( Fig. 50 ) increased in freq uency to 11-20 cells per 2 x lOs JL2 and it expa nded fu rt he r iOlo arcus latera l to the [lAG and dorsal to the nucleus cuneiformis. In contrast to these striking increases 24 days after inoculation, the lale ral PAG is nm markedly different from the control and 4-day post-inoculatio n animals. Aft er approximately 17 days o f arth ritic pain. however, the ven tromed ial PAG and dorsal ru phc contained more hybridization-positive neurons than a l the two earlie r

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Fig. S. Maps of rCjl rc;,cntnllve hybrid I7.a tion-posihve neuron frequenci<:s II I the level of the ell udal one- third of the I'AG 11\ pol}1lrthritlc r~t~ (Sprall u.:- Dawley Strum, II - 8). A: oonL rul 1ll1imilis received injections of mll1erlll oil ~ehicle . No sLlb~tn n tinl diffe rences were observed at llOnl cun trols front each of the tim e ",H nls. U: POIYlltlh ru ic animal" duys afte r inocui:l ti()n. C: polYDrthritic ammal 7 d:IYs after inoculatiun. I): polyart hrilic annllul 24 li:lyS ufter inocu lat illn. DR .. dorsal ra phe nuc le us: I'AG - pe riaqued uctlll limy; CuF .. nucle us cunciformis; SCI' supe rior cerchell:tr pcliullcte; r NO - ponti ne reticulilf nucle us ma lis: MiTII - tIl lcmccllulnr tegmc nlal nucle us.

94

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4 Days

Control

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Fill. 6. Map) of rcprcsc nmtillc hybrj d i~!l t jo n . po!Ii t i\le neuron frequ encies HI lhe level of th e middle one-third of Ihe r AG in polY;lnhri lic ra tS (Sprng ue- I);lwley Simin, /I '" 8). A: cO ll troillllimals received inj ections of min eral oil vchicle. No SubSllHitial differc llCcs were obserwd amonS co mTois from cl!ch of the lime points. B: polyarlh ri lie ;Inimlll " days aft er illocul~lion . C: polyarthrit ie anim" 1 7 days after inoculnt ion. 0 : polYllrlh r jlic lU1Imrii 24 days aner in ocul atio n. RN '" red nucleus: PAG ... pe riaqucdnctnl gray; SC ... superior rolJicul is: ML ... medial lemniscUS: DpM e ... deep meSCnCCI)hltlie nuclei.

li me poin ts. Thus, with in the ca udal third of the I' AG , Ihe onse t of polya rth rit is resulted in a modest increase in the frequency of neurotensin mR NA-containing neurons. Increases in neurotensin mR NA expression due to chronic polya rthritis we re most apparent out side of the PAG, in the nucleus cuneiform is and the lateral tegmental areas. At more rostral midbrain si les. incl udi ng the middle

Control

of t he PAG, a similar pallern of arthritis-induced effects was observed. Four days following inoculation (Fig. 6B). the distribution and frequency of neurOlensin hybridization-positive neurons was practica lly ind istinguisha ble from the controls ( Fig. 6A). Seven dllYS following inocul ation, coi ncident with the onset of pain. increased neuron frequencies were observed in the ve ntral PAG and the dorsa l raphe (Fig. 6C). A larger

1 day post-injection 6' 10~ISl2x lcr~2

17 day ligatu re

16 day post-in jection _

11 '20 celis/2Xl0S ~

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>20 C811912x\05 ~2

Fig. 7. MUI)) of reprc.>cfltll live hybridil.D lion·posilive neuron frequ encies UI Ihe level of Ihe cu udul one·lhird of Ihe r AO in fillS wilh unilulentl nocice plt ve lesio ns (Sprugue- Dawtey stra in. 11 - 6). A: w nlrol n n i m u l ~ received pl nntnr injections of mineral oil vehicle. No s u bsl n nl i ~1 diffe re nce~ we re ob~erved umon ll con l m l ~ from e.tch of th e time pomls. II: uni llUernl l)ilW inn n mm ~ l ion bnimlll I dny aft er inoc ulrol ion. C: unil ll lc rnl paw i nn ~ mrn ll l i(>n anim ul 1(, d:lYs Itfter inocul plio u. D ~ peripheral rnOllOne uropnth y lmimnl 17 dnys afl er schtlk lignt inn. DR - d OfS ld ra phe nucleus; I'AO - peri nqueducluJ lI ray: CnF - n u cle u ~ cu ncifomlis: SCI' - superior cc rcbe llor peduncle; I' NO - po nline rc tieulltr nucleus notlis: MiTII - microcellular leg memrol nucleus.

\

95 ron frequency were observed in the lateral PAG , the ven trolate ral PAG , o r the adjacent nucleus cuneiform is. In contrast 10 the modest early effects. grea ter increases in neuron frequency were evident in the nu· cleus cuneiformis nnd ventral PAG sixteen d:.ys afler unilnteral adjuvant injection (Fig. 7C). In animals that hnd sciatic ligatures, greater increases above normal neuron frequencies were evident in Ihe caudal midbrain. Seventeen days after liga tion. the ventrolateral PAG, the nucleus euneiformis, nnd the lateral tegmentnl nuclei all exhibit increased frequencies of hybridiz.1tion-positive neurons contralate ral to the lesion . In addition, the tegmen tal areas in which hybridizntion·positive neurons were found had expanded. In both th e unilateral in nammation and sciatic ligature models (Fig. 7C,D), increased frequencies of hybridization-positive neurons were also noted in the dorsolateral portion of the orall>ontine reticul ar nucleus, ventral to the nucleus cune ifo rm is. At the level of the mid-PAG, alterations in the distributions and frequencies of neurotensin hybridization-positive neurons were not as great as those observed in the polyarthritic :tn imals. One day after unilateral paw injection of adjuvant (Fig. 8B), the ventral I'AG and dors:.tl raphe were the only regions in which neuron frequencies increased above control levels (Fig. 8A). Sixteen days afte r injection with adjuvant (Fig. SC), hybridization-po!>itive neurons were found in the deep mesencephalic nUclei, but were less freque nt in the dors:.1 raphe nucleus and ventra l I'AG. A simi lllT p:lllern of ncurotensi n milNA expression was evident

area of the lateral PAG contai ned hybridization·positive neurons, whieh also appear within the deep mesencephalic nuclei. Venlral 10 the PAG, neurOiensin mRNA expression was seen in midline areas between the red nuclei. As Ihe early symptoms became chronic, 24 days after inocu lation (Fig. 60), the frequency of hybridization-positive neurons in the dorsal raphe, the surrounding PAG, and the midline areas between the red nuclei diminished but remained grealer than control levels. At the s:tme time, increases in the frequency of hybrid ization-posi tive neurons were evident in tegmental regions laleral to the PAG. Unilateral paw ;'1/1a",flltllioll and .fcimic ligall/res.

The effects of paw innammation or sciatic ligation on the distribution patterns and frequencies of neurotensin hybridiza tion-positive neurons arc similar to Ihose observed in the polyarthritis model. Although bilateral changes were observed. we have mapped the midbrain contralateral to the lesion because the effects Were more consistent and more striking. At the level of the caudal one third of the PAG (Fig. 7), the effects of unilateral adjuvant injection (Fig. 7B and 7C) are comPared to animals that received mineral oi l vehicle alone (Fig. 7A). Hybridization patterns and frequencies in the mineral oil-i njected controls were practiclllly iden· tical to those observed in normal, uninjected ra ts (data not shown). One day after adjuva nl injection into onc hind paw (Fig. 7B), the grea test changes were observed in Ihe dorsal rnphe nucleus and Ihe adjacent ventromedial PAG . The size of the high frequency ce ll group increased ven trally nnd talerolly. No increases in neu·

Control

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FiR. 8. Maps of reprc.scn t:lllvC h~hridllJl1 iun ·poshive neuron frequencies III Ihe tevel of Ihe middle one·third ur Ihe PAG in mls wi,h unihuerat nociceplive Icsions (Spfllguc- I)uwley Slmln. ,, - 6). A: oonlroJ animnls received plion1:lr lJ1jeetinns of mineml oil vehicle. Nu subs,:ul1il!1 differcnees were observed llmoni oomrots frum each of th e lime poinls. U: uuilalcrul paw inflammatiOn auimlll I day after inoculation. C: uuilHlcml pllW inflammilium nnimul 16 d :IY~ after inocu tntion. D: peripheral mouou eurOIl:uhy QIIIIIlOI 17 dllYs [Ifler ~iali c liau[ion. RN " Red Nuclcu~: I'AG - I'crinqueduellil Gmy: SC .. Superior Coll iculis: ML .. Mediat L..emni~eu ~: t)pMe - deep IIlese ncep halic nuclei.

96 17 days after sciatic nerve ligation. Frequencies of hybridization-positive neurons increased in the deep mesencephalic nuclei and the ventral PAG, and larger areas of the deep mesencephalic nuclei contained hybridization-positive neurons. In addition, the red nuclei and regions medial to the red nuclei showed increased frequencies of neurotensin mRNA-containing neurons after 16-17 days (Fig. 8C,D). Summary of hybridization histochemical data We employed three different experimental models of chronic nociception to permit more generalized conclusions about the influence of that nociception on the anatomical patterns and frequencies of neurotensin mRNA expression in the midbrain. Three general observations stand out. These findings are summarized in Table I, which lists the cell frequencies and changes in the area of cell groups that were depicted in Figs. 4 through 8. First, the table excludes the midbrain at the level of the rostral one third of the PAG. This region was examined in all three models using hybridization histochemistry. Among all sections from the rostral 1/3 of the PAG, no consistent alterations in neurotensin mRNA were found (data not shown). Second, at the level of the middle and caudal one third of the PAG, the early effects of the lesions produced by Complete Freund's Adjuvant are confined to the ventral PAG and the dorsal raphe. Figs. 5C, 6B, 7C, and 8B all demonstrate increases in either the frequency or field area of neurons that are hybridization-positive for neurotensin mRNA. Within the middle third of the PAG, the early effects were diminished, but remained above control levels, when the nociceptive stimulus became chronic (Figs. 6D vs. 6C and 8C vs. 8B). Third, chronic nociceptive stimuli lasting 16-17 days produced the most striking increases in neurotensin mRNA expression within the nucleus cuneiformis, the lateral tegmental nuclei (including the pedunculopontine and microcellular tegmental nuclei), and the deep mesencephalic nuclei (Figs. 4B, 5D, 6D, 7C,D and 8C,D).

Thus, acute nociception affected neurotensin mRNAcontaining cells in the medial portion of the midbrain. As the nociception became more chronic, effects were observed in the lateral regions of the midbrain. Quantitative analysis of hybridization- and immunohistochemistry in the midbrain Hybridizations. The anatomical mapping studies presented above describe populations of neurons in which the de. novo transcription of detectable levels of neurotensin mRNA is apparently induced by the nociceptive stimuli. However, the data do not address the question of whether the pain might also modulate neurotensin mRNA levels within neurons in which it was already transcribed. The peripheral neuropathy animal model was chosen for this analysis. We employed quantitative image analysis within 8 discreet regions of the caudal midbrain (Fig. 9A) on cryostat sections from animals that received 17 days of sciatiC ligation to analyze cellular levels of neurotensin mRNA Midbrain regions that were ipsilateral to the ligated sciatic nerve were used to control for individual variations in fluorescence intensity, frequency, and distribution of the hybridization-positive neurons. Areas 1 and 2 are immediately subjacent to the aqueduct in the ventral PAG. Areas 3 and 4 are at the ventrolateral edge of the PAG, areas 5 and 6 are within the nucleus cuneiformis, and areas 7 and 8 are in the lateral tegmentum. Within 1.6 X lOs JLm 2 fields, significant differences were observed between the average neuronal fluorescent signal strengths from the contralateral and ipsilateral sides of the midbrain. Fig. 9B graphs the calibrated average neuronal gray levels from video micrographs of neurons in the paired sampling regions (Fig. 9A). The neurons that were quantitated were selected by the image analysis system based on a combination of morphological criteria and the presence of a fluorescent signal. Among those neurons, statistically significant increases ranging from 28 to 39% were found in the fluorescence intensity. Thus,

TABLE I Summary of neuron frequencies (as gray levels 0 through 4) taken from each of the representative maps in Figs. 5 through 8

Pll!s signs indicate that the area occupied by the hybridization-positive neurons at the listed location had increased in size. Paw injection

Polyarthritis

Ventrolateral PAG-caudal Nucleus cuneiformis Microcellular tegmentum Dorsal raphe - caudal Ventral PAG - mid Deep mesencephalic N. Dorsal raphe - Mid

Sciatic ligation

Cont.

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7 days

24 days

I day

16 days

17 days

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Ihe unil ateral sciatic liga tions caused cellul ar ne u· rotensin mRNA levels to increase contrahlle ral to the SCiatic ligation . Beca use the average signal intensi ty increased above control leve ls, neurotensin mR NA lev· cis appear to have increased within neurons that nor· Illally contain the message. Immullohistochemjstry . It was importan t to determine whether the observed inerctlses in ne uronal ne u· rotensin mRNA were accom panied by increases in neu rote nsin pe ptide leve ls. We examined the midbrain regions depicted in Fig. 9A using immunohistochem· istry with avidin·biotin DAB vis ua lization. Immuno· staining in the regions shown in Fig. 9A (depicted in Fig. 2) consisted predom inantly of punctate terminals and occasional densely.stained fibe rs. Imm unoreactive Pcri ka ryll were lightly stained and best demonstra ted With the aid of contrast·e nhanced video microscopy. In Video micrographs having 4 X 10 4 J.l-m2 fields, we se· lected for tcrminals and fibe rs based on their morphol· ogy and their staining intensi ty, and Ihe n quantitat ed the area of the se lected structures. As demonstrated in Fig. 9C, a medial to lateral grad ient in the area of illlrnunoreactive termina ls and fibers existed tlmong the quan tit:lled regions. Interestingly, both the ventrolat · eral PAG and the nucleus cuneiformis contained a Significan tly grea ter area of ne urotensin·imnmno reac· tive tcrmina ls and fibe rs on Ihe side contralateral to the sciatic ligatures (5 1% a nd 166% increascs. respec· tively). The obscrvation that fewer immunoreactive termi· nals nnd fibers we re presc nt in the nucleus cune iformis and latera l tegmental arcus than in the PAG is can· firmed in low powc r micrographs of ne urotensi n 1m· nlllnostai ning in the midbrai ns of an imals havi ng uni· Intcrnl ne uropathies. At the rostrocauda l leve l of the mid· I'AG (Fig. lOA), sligh tly grea ter staining is visible On the left half of the I'AG (arrowhead) than on the right side. At the level of the caudal one· third of thc PAG (Fig. lOB), typiclll of thc sections quantit atcd for Fig. 9, marked incrcases in neu rOlensin immunoreactiv· ity :Irc visiblc in the vc nt rolatcral PAG o n thc left side (contralatera l to the lesions) and the staining extends into the nucleus cu neiformis (arrowheads). These reo sults suggest that increases in neurole nsin mRNA are, indeed, accompnn ied by increased production of neu· rOl ensin peptide in the sciatic ligation model for painful pe ripheral neuropathy. DISCUSSION Tec/".iclIl cQllsitJer{l/i01lS

The int cnsity of our nuoresce nt hybridiza tion signal is improved ove r II prcvious report 4.1 in which biotiny.

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Sampling Region Fill· 9. Image IIn n lysi~ of ncurotcnsi n 1IlRNA h~lm (h7.atlon III ~to. chcmistry Hntl neuTIlt ensi n im1lluno histochemi,try in th e ca ud ul mid· brain of IInimals ha vi n, lIIulaterul ~riphcra. 1 nCllrop:ulues. A: mid. brain sites at which 4 X 10 4 ~m J fie lds .... ere unll l)'SI,:d. Sites I. J. S :lIld 7 were ipSll:uerat to Ihe scmllC haa t lon ~ 8 : ;lVeraae neuronlll Ilra), le\le l5 frOIll each of the SII C3; dl.! plcted m p;lnel (A). Ne l.l ro n~ withm tac h rlCld ..,.. ere automlllicOilly se tectcd for II nat)'Sls based nn !I,lnal intensity and morphologica l criteria. ('allbruted 8m)' le~d~ MC based on a curve in which n standa rd fluoresce nce intc ns,ty 15 Hssill nl.!d an lututMIlY vnl uc of 4, as dCM: ribcd 111 M n t erilll~ and Methods. Significam differe nces llmo ni puired unalysis si t e~. tl~ de termined by ANOVA, Ire dcsianat ed by aste risks (n " 4) C: thl.! tlensit)' of ncurotcnsi n· immunorcllctlVe tc: nmnuls was determmed by electronically removi nll hllh ter·stalnlnll ptnluT)-a. and quantltatlnll the area of Slructures h!wlIl, the size, shape: and stalllllll1lll tensity of alon termmals. Data Ire reprcsc:n' ed as the: area of Immunorellctl\'C structures thM me l these criteril\ per 4 x 1O~ "n,2 ficld. Sijj:nincllnt dlffercnce:> limon, p:lirell .mlllysis silcs. liS determ Ined by ANOV A. nre deslgn~l l.!d hy :1 ~ l c risks (II " .1).

98 laled DNA oligonucleotide probes were also used. The improvements ,Ife partinlly allributablc to increased efficiency of the oligonucleotide biotinylation renction , This was achieved by simply decreasing oligonucleotide concentrations in relation to the other reactants and by adding additional Icrminal Iransfcrasc halfway through the reaction pcricxi. Additional improvements in hybridization sensitivity were obtained by using video microscopy. The sensitivity of nuorcsccnce detection was gfC,lIly enhanced by perrorming electronic contrust-e nhancement of the video images. In addition, it was possible to visualize perikaryal immunoreactive ncurotcnsi n wi thout colchicine. which may alie f rates of peptide biosynthcsis I5•32•4II • We obscrved neurotensin immunoreactive I>erikllrya in ellch of the midbrain regions in wh ich hybridization-positive perikal)'u were found . Because the pcrikal)'al immunoreactiyity was light. they were easily excluded from quantilatiye analyses of terminal and fiber distributions. The form of the maps in Figs. 4 through 8 is one we prefer when comparing large amounts of anatomica l data. Our hybridizations hay, ide ntified many Ihous..1 nds of neurons contnining neurotensin mRNA. Unlike camera lucida drawings. these maps arc made without exhaustively recording the ex .. cl local ion of each hybridization-positive neuron and anatomicnl landmark . In!>tead. neuron frequ encies (cells per unil area) are plolled against major landmarks as depicted in a standardized rat brain atlns)'}, This method has two advllntugcs: the dnta are easy to interpret because the format is standardized and the maps nrc much eusier to make. One disadvantage of the method is thai

some resolution is lost by lumping the neuron frcquencies into four groups. Some real differences might be emphasized while others could be diminished. Theoretically, a difference between 9 and II cells/2 x lOs l could resemble the difference between 6 and 20 cells/2 X lOs 1-'2. Among the examples we chose fOf Figs. 4 through 8, the on ly frequency difference Ihat we be lieyc is exaggeratcd occurs in the dorsal raphe nu' cleus of panels A and B from Fig. 5. Four days after adjuvant inoculation, distributions of hybridizationpositive neurons in the dorsal raphe of polyarthritic anima ls we re generally similar to vehicle-injected controls. Finally, in areas where steep grad ients in neu ron frequencies were encounte red (such as nea r the dorsal raphe nucleus, where gray level 4 was adjacen t to gray level 2), we did not include narrow bands of intermedi· ule density because the d iffe rences in gray scales arc difficult to resolve.

Rel(llionship bellVeell the iI/tensity of the I/ociceptil:e stimuli alUl the IIUlgllitlllle of 'he challges observed by ill siw hybridizatioll The present data confirm Ihal sources of nociceplion having different etiologies all produce similar re' suits on neuronal neurotensin mRNA levels in the midbrain (l nd, presumably, also on neurotensin peptide levels, In addition, the present studies allow us to qua liHlIiyely compare the experimental models and correlate patterns of neuronal mRNA expression with Ihe magnitude of Ihe nOCiceptive stimulus. Unfortu· nately, the preferred behavioral parameter in the unilateral models - differences in paw withdrawal latcn·

Fig. 10. Cont r/l)t-c nhan ced video microafaphs of neurolcnsin immunohislochemistry In the cll ud r,1 midbfllin of animals havina unilutcral pc:riphcflll ncuropl,thie,. A: 01 Ihc level of the mid I'AG, ~lIlh tl y morc IIllnlUnorcl'l.:livity is VIsible on the side oonlmlUlerol 10 Ihe les,on (arrowhead), U: al the level of Ihe caudal olle third of Ihe rAG , Ii,rlc inereuses in neurol cnsin ,mmllllOSluininll were upparenl in Ihe ventrllt ol.d IlIlerlll I'AO IUld 111 the nucleus eun ciformis (lirrowheods) t'Onl rllliu eral lo the .,,:illtie I1l1olio,,~. Scale b~f - I mill.

99 cies from noxious radiant heat- cannot be applied to bilaterally-inflamed polyarthritic animals. Effects on this modality, preferred for the peripheral mononeuropathy model, has not been reported in the. polyarthritic model 17 • A priori, we believe that adjuvant-induced polyarthritis causes more pain than either of the unilateral models because the lesions are bilateral and may involve the tail, hip and lumbar vertebral articulations in addition to more distal joints. Behavioral observations support this assumption. Neither the animals that received unilateral adjuvant inject,ions nor the animals that developed unilateral peripheral neuropathies ever vocalized upon handling, failed to gain weight, or were observed to hyperventilate. In the polyarthritic model, Wistar Strain animals exhibited greater morbidity than the Sprague-Dawley Strain. Between the models employing unilateral lesions, paw Withdrawal latency difference scores (Fig. 1) suggest that the early effects of adjuvant-induced inflammation Produced stronger hyperalgesia than the early effects of sciatic ligations. The rank of the paw withdrawal difference scores reversed after the lesions become chronic. Interestingly, neurotensin mRNA-containing neurons were found in the red nuclei of the unilateral chronic pain models, but not in the red nuclei of polyarthritic animals. This coincidence may be related to the paw guarding posture typical of both unilateral models but not the bilateral polyarthritis model. Based on this general comparison of behavioral data, We believe that the order of severity for the models of chronic nociception used in these studies is: polyarthritis - Wistar > polyarthritis - Sprague-Dawley> peripheral mononeuropathy > unilateral adjuvant inflammation. In light of this rank order, we examined the frequencies of neurotensin mRNA-containing neurons in various areas of the caudal midbrain in search of a correlation to the severity of the nociceptive lesion. Simple inspection of the maps presented in Figs. 4-8 and summarized in Table I show that the frequencies of hybridization-positive neurons correlate best With the rank order of behavioral symptom 'strength in nucleus cuneiformis and the lateral tegmental areas. The notable exception is the peripheral mononeuropathy model, in which the nucleus cuneiformis and lateral tegmental areas contain hybridization-positive neuron frequencies that exceed those found in the polyarthritic Sprague-Dawley Strain animals. Notwithstanding the unexpectedly high frequency of neurons in this one model, we believe that peptide expression in the anatomical areas lateral to the PAG (nucleus cuneiformis, microcellular tegmental nucleus, and deep mesencephalic nucleus) may be a unique feature of

chronic nociception. The significance of increased neurotensin expression lateral to the PAG is currently unclear because the circuitry involving the neurotensinergic neurons has not been established. However, recent studies have indicated that collaterals of spinodiencephalic fibers terminate in the region of the microcellular tegmental nucleus and these fibers are reported to transmit a variety of somatosensory information, including nociception 12,13,16. Since our hybridization data indicate that modifications in the neurochemistry of the microcellular tegmental nucleus is correlated with exposure to chronic nociception, it is conceivable that the microcellular tegmental nucleus and areas immediately dorsal to it are involved in processing nociceptive information. Further studies are underway to investigate whether this area facilitates or inhibits ascending transmission of chronic nociceptive stimuli. Regions involved in pain-related alterations of neurotensin mRNA levels It was not surprising to find that pain modulates neurotensin mRNA levels in the PAG. The PAG is uniquely situated to play an important role in descending antinociception 4 ,47, because of its spinal and telencephalic afferents combined with efferents to ventral medullary regions that are critical· to descending antinociception. Early evidence indicated that one of the major functional roles of the PAG was antinociception and that this function was mediated largely by projections from the PAG to the ventromedial medulla 4 ,9,34,35,47. Recent studies indicate that antinociception is but one of many important roles played by the PAG, Emerging evidence suggests that the PAG may serve to integrate the behavioral, cardiovascular, and respiratory responses to threatening or stressful situations, in addition to modulating nociception. In general, the PAG seems to integrate components of coordinated responses necessary for the animal's survival (reviewed in ref. 3). The dorsal raphe nucleus, surrounded by the ventral PAG, has been recognized as a site from which stimulation-produced analgesia could be obtained without eliciting behavioral side effects 22 • In addition to providing a major serotonergic projection to the diencephalon, the dorsal raphe nucleus also projects to the rostroventral medulla, including the nucleus raphe magnus. In particular, descending projections originating in the lateral wings of the dorsal raphe nucleus have been identified in retrograde tracing studies 21 ,46. It remains to be demonstrated whether the neurotensin mRNA-containing neurons in the dorsal raphe nucleus project to the ventral medulla. On the other

100

hand, a significant number of PAG neurotensin containing neur
may occur through pathways other than the lateral spinothalamic tract - the major pathway for nocicep' tive .information - is reviewed by Guilbaud 25. The possibility that alterations in neuronal neurotens in mRNA occur in ascending nociceptive pathways can' not be discounted. However, the potent antinociceptive actions of exogenous neurotensin 1•30 suggest that the activated neurons may have a role in antinociception. Based on our present understanding of antinociceptive systems, this implies that descending pathways are involved. The present data, from three different a~imal models, suggest that the early effects of nociception in the midbrain differ from the later effects. The early increases in both the frequency of hybridization-positive neurons and in perikaryal levels of neurotensin mRNA occur in the ventral and ventrolateral PAG and the dorsal raphe nucleus. Later, after approximately 17 days of nociceptive input, changes were observed in the lateral tegmental areas and nucleus cuneiformis in addition to the ventrolateral PAG. An understanding of that basic difference, whether it involves the potentiation of quiescent circuits, the recruitment of pathwayS not normally associated with nociceptive transmission (as suggested by Guilbaud 25 ), or the sensitivity of individual neurons to peptide gene induction, could help elucidate the qualitative differences between chronic and acute pain.

Conclusion In three different models for chronic nociception, we have observed that neurotensin mRNA levels are increased in midbrain areas where it is normally found. Neurotensin mRNA expression is also induced to detectable levels in neurons in which it is not normaIly observed. The ventral periaqueductal gray, including the dorsal raphe nucleus, are affected soon after the onset of nociception. In contrast, neurons in the nucleus cuneiformis and lateral tegmental regions respond more slowly, but maintain neurotensin mRNA levels that are higher than normal. In addition, the frequencies of hybridization-positive neurons in these late-responding areas correlate with behavioral criteria that are thought to indicate the strength of the nociceptive lesions. Increased neurotensin peptide levels accompany the increases in mRNA, suggesting that neurotensin in the midbrain may playa role in modulating acute or chronic nociceptive stimuli. The present finding that chronic nociception results in increased neurotensin mRNA levels and increased immunoreactive neurotensin peptide levels underscores the importance of elucidating the pathways through which neurotensin produces antinociception.

101 Acknowledgements. This research was supported by NIH grants NS 28016 to FGW and DE 06682 and DA 06687 to AJB. The authors thank Lorraine Wellman for her excellent technical assistance.

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