increased uptake and transport of cholera toxin b-subunit in dorsal root ganglion neurons after...

Increased Uptake and Transport ofCholera Toxin B-Subunit in Dorsal Root

Ganglion Neurons After PeripheralAxotomy: Possible Implications

for Sensory Sprouting

YONG-GUANG TONG,1 H. FREDRIK WANG,2 GONG JU,1 GUNNAR GRANT,2

TOMAS HOKFELT,2* AND XU ZHANG1,2

1Department of Neurobiology, Institute of Neurosciences, The Fourth Military MedicalUniversity, Xi’an, People’s Republic of China

2Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

ABSTRACTIn the present study we show that, in contrast to the rat, injection of cholera toxin

B-subunit (CTB) into the intact sciatic nerve of Macaca mulatta monkey gives rise to labellingof a sparse network of fibers in laminae I–II of spinal cord and of some mainly small dorsal rootganglion (DRG) neurons. Twenty days after sciatic nerve cut, the percentage of CTB-positivelumbar 5 (L5) DRG neuron profiles increased from 11% to 73% of all profiles. In the spinalcord, a marked increase in CTB labelling was seen in laminae I, II, and the dorsal part oflamina III. In the rat L5 DRGs, 18 days after sciatic nerve cut, the percentage of CTB- andCTB conjugated to horseradish peroxidase (HRP)-labelled neuron profiles increased from 45%to 81%, and from 54% to 87% of all neuron profiles, respectively. Cell size measurements in therat showed that most of the CTB-positive neuron profiles were small in size after axotomy,whereas most were large in intact DRGs. In the rat spinal dorsal horn, a dense network ofCTB-positive fibers covered the whole dorsal horn on the axotomized side, whereas CTB-labelled fibers were mainly seen in laminae III and deeper laminae on the contralateral side. Amarked increase in CTB-positive fibers was also seen in the gracile nucleus. The present studyshows that in both monkey and rat DRGs, a subpopulation of mainly small neurons acquiresthe capacity to take up CTB/CTB-HRP after axotomy, a capacity normally not associated withthese DRG neurons. These neurons may transganglionically transport CTB and CTB-HRP.Thus, after peripheral axotomy, CTB and CTB-HRP are markers not only for large but also forsmall DRG neurons and, thus, possibly also for both myelinated and unmyelinated primaryafferents in the spinal dorsal horn. These findings may lead to a reevaluation of the concept ofsprouting, considered to take place in the dorsal horn after peripheral nerve injury. J. Comp.Neurol. 404:143–158, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: plasticity; spinal cord; pain; nerve injury; GM1 monoganglioside; DRG

Dorsal root ganglion (DRG) neurons of the rat can bedivided into two main categories, i.e., large and smallneurons, among which the large neurons give rise to thickmyelinated axons and the small ones to unmyelinatedaxons (afferent C fibers) as well as thin myelinated axons(afferent Ad fibers) (see Willis and Coggeshall, 1991). Theunmyelinated nociceptive primary afferents terminate inlaminae I and II of the dorsal horn of the spinal cord, thethin myelinated mechano-nociceptive afferents in laminaeI and V, and the thick, myelinated afferents in laminae III

Grant sponsor: Nature Science Foundation of China; Grant numbers:39525010, 39500045; Grant sponsor: Swedish Medical Research Council;Grant numbers: 2887, 10820; Grant sponsor: Marianne and MarcusWallenberg’s Foundation; Grant sponsor: Astra Pain Control AB; Grantsponsor: Royal Swedish Academy of Sciences.

Drs. Tong and Wang have contributed equally to this study.*Correspondence to: Tomas Hokfelt, Department of Neuroscience, Karo-

linska Institutet, S-171 77, Stockholm, Sweden.E-mail: [email protected]

Received 6 August 1998; Revised 16 October 1998; Accepted 28 October1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 404:143–158 (1999)

r 1999 WILEY-LISS, INC.

and IV (see Maxwell and Rethelyi, 1987; Willis andCoggeshall, 1991; Grant, 1993, 1995).

Peripheral nerve injury in the rat has been reported toinduce sprouting of myelinated primary afferent fibersfrom deeper laminae into lamina II of the spinal cord, andit has been suggested that such a structural reorganiza-tion may contribute to the development of chronic painmediated by large myelinated primary afferent fibers aftera peripheral lesion (Woolf et al., 1992, 1995; Woolf andDoubell, 1994; Bennett et al., 1996; Lekan et al., 1996;Mannion et al., 1996, 1998; Coggeshall et al., 1997; Doubellet al., 1997; Doubell and Woolf, 1997). Evidence for sprout-ing of primary afferent fibers has also been reported instudies on monkey spinal cord after peripheral nerveinjury (Florence et al., 1993).

The demonstration of sprouting of large primary affer-ent fibers is in almost all studies mentioned above basedon two retrograde and transganglionic neuronal tracers,the B subunit of cholera toxin (CTB) and CTB conjugatedto horseradish peroxidase (CTB-HRP). Cholera toxin wasoriginally introduced as a retrogradely transported markerby Stoeckel et al. (1977). This bacterial (Vibrio cholerae)toxin binds to cell membrane glycoconjugates, especially tothe monoganglioside GM1 (Cuatrecacas, 1973; Holmgrenet al., 1973; King and van Heyningen, 1973). The bindingsite of the cholera toxin molecule is located at its Bsubunit, also called choleragenoid, which is responsible forthe uptake characteristics of this toxin (Stoeckel et al.,1977; Dumas et al., 1979). CTB and CTB-HRP have beenused as anterograde and transganglionic tracers to demon-strate the central terminal fields of primary afferents formany years (Trojanowski et al., 1981; Wan et al., 1982a,b;Robertson and Grant, 1985; Lindh et al., 1989; Rivero-Melian and Grant, 1990a,b, 1991; LaMotte et al., 1991;Rivero-Melian et al., 1992). These studies show that, in therat, CTB and CTB-HRP are mainly taken up by the largeDRG neurons that under normal circumstances, as men-tioned above, project into laminae III–IV of the spinal cord.

Peripheral nerve injury induces marked changes in theexpression of neuropeptides, neuropeptide receptors, andnitric oxide synthase in DRG neurons of the rat andmonkey, resulting in a virtually new phenotype of the DRGneurons with regard to these molecules (see Hokfelt et al.,1994, 1997; Zigmond et al., 1996). Peripheral axotomy isaccompanied not only by alterations of messenger mol-ecules and their receptors, but also by a change in bindingof plant lectins. For instance, the isolectin B4 of the plantlectin Griffonia simplicifolia I (B4) binds to a subtype ofsmall DRG neurons (Streit et al., 1985, 1986; Silvermanand Kruger, 1988, 1990; Wang et al., 1994), and afterperipheral nerve injury, a reduction in the lectin B4binding was observed in the superficial dorsal horn of ratspinal cord (Molander et al., 1996).

The primary aim of the present study was to character-ize further the possible sprouting process in the monkeyspinal cord after peripheral nerve injury (cf. Florence etal., 1993). In view of the surprising results we alsoincluded, for a direct comparison, peripherally axotomizedrats, which previously have been studied in much greaterdetail with regard to sprouting (Woolf et al., 1992, 1995;Woolf and Doubell, 1994; Doubell et al., 1997; Mannion etal., 1998). CTB and CTB-HRP (only in rat) were used as aretrograde and transganglionic tracer, and spinal cord,lower medulla, and DRGs were examined in monkey and

rat after complete unilateral sciatic nerve transection(SNT). The very robust and dramatic effects observed inthe monkey less than 3 weeks after axotomy, led us to usesimple profile counts for the quantitative analysis through-out the study (see Discussion section).

MATERIAL AND METHODS

Nerve transection and tracer injection

Fifteen adult male Sprague Dawley rats (ALAB, Stock-holm, Sweden), each weighing 200–250 g, and six adultmale Macaca mulatta monkeys (Institute of Neuroscience,Xi’an, The People’s Republic of China), each weighing 3–4kg, were used in the present study. All efforts were made tominimize animal suffering and to use as small a number ofanimals as possible. The experiments were carried out inaccordance with the policy of the Society for Neuroscienceon the use of animals in neuroscience research, andapproved by the Stockholm local animal ethical commit-tee, and the Chinese National Committee of the Experimen-tal Animals for Medical Purposes, Shanxi Branch, PeoplesRepublic of China.

Under deep anaesthesia, by intraperitoneal injection ofchloral hydrate (300 mg/kg) for the rat and sodium pento-barbital (70 mg/kg) for the monkey, the left sciatic nervewas exposed and transected at midthigh level (nine ratsand six monkeys). A portion of the proximal part of thetransected nerve was resected (5 mm for the rat, 10 mm forthe monkey). The wound was closed, and the operatedanimals remained in a standard environment at theanimal departments for the postoperative period.

The rats were, after postlesion times of 14 days (n 5 6)and 122 days (n 5 3), anaesthetized as above, and thesciatic nerves on both sides were exposed at the midthighlevel. Two microliters of 1% CTB (List Biological Laborato-ries, Inc., Campbell, CA; three rats at each postlesion time)or 0.5% CTB-HRP (List; three rats for postlesion time 14days, only) dissolved in 0.1% Fast Green in distilled water,were slowly injected with a 33-gauge Hamilton syringeinto the proximal part of the transected left sciatic nerve(about 4 mm proximal to the stump) and into the rightintact nerve at the same level. The injection was also madeinto the right sciatic nerve of six other, unoperated rats(three rats for CTB, three rats for CTB-HRP) as control.

The monkeys were deeply anaesthetized after postlesiontimes of 14 (n 5 3) and 90 (n 5 3) days, and the sciaticnerves were exposed bilaterally. One hundred microlitersof 1% CTB (List) was slowly injected with a 16-gaugeHamilton syringe into the proximal part of the transectednerve (about 7 mm proximal to the stump) and into theright intact nerve at the same level.

Fixation and tissue preparation

After tracer injection (4 days for rats and 6 days formonkeys), the animals were deeply anesthetized as de-scribed above, and perfused via the ascending aorta withwarm (37°C) saline (50 ml for the rat and 1,000 ml for themonkey) followed by a warm solution composed of 1%paraformaldehyde and 1.5% glutaraldehyde in 0.1 Mphosphate buffer (PB) at pH 7.4 for CTB-HRP–injectedrats, or by warm solution consisting of 4% paraformalde-hyde and 0.2% picric acid in 0.16 M PB at pH 6.9 forCTB-injected animals (150 ml for the rat and 1,000 ml forthe monkey). The perfusion was then immediately fol-

144 Y.-G. TONG ET AL.

lowed by the respective, but ice-cold, fixative for another15 minutes (400 ml for the rat and 2,000 ml for themonkey). The brainstem, lumbar spinal cord, and L5DRGs were dissected out, postfixed in the same fixative for90 minutes at 4°C, and were then immersed in 10%sucrose in 0.01 M phosphate buffered saline (PBS) over-night. For CTB-HRP–injected rats, 40-µm-thick trans-verse sections of the caudal medulla and lumbar spinalcord segments and 14-µm-thick longitudinal sections ofthe DRGs were cut in series on a freezing microtome andkept free-floating in PBS. For all CTB-injected cases,20-µm-thick transverse sections from the caudal medullaand lumbar spinal cord segments and 14-µm-thick longitu-dinal sections from the DRGs were cut in series on acryostat (Microm, Heidelberg, Germany), and mounted ongelatin-coated slides.

HRP histochemistry

The free-floating sections were processed for HRP histo-chemistry by using tetramethylbenzidine as the chromo-gen and stabilized with sodium nitroprusside according toMesulam and Brushart (1979). The sections were thenair-dried overnight, cleared in xylene, coverslipped withEntellan mounting medium (Merck, Darmstadt, Ger-many), and examined in a Nikon Microphot-FX microscopeunder brightfield illumination.

CTB immunohistochemistry

The slide-mounted sections were processed either byindirect immunofluorescence or by immunoperoxidase his-tochemistry. The incubations were carried out in a humidchamber and at room temperature, except for the incuba-tion with primary antisera at 4°C. All antisera used werediluted in PBS with 1% bovine serum albumin (BSA) and0.3% Triton X-100 (TX) added. Briefly, the sections werepreincubated in BSA-TX for 1 hour, and incubated over-night with a goat antiserum against CTB (1:1,000; List).After several rinses in PBS, the sections were incubatedfor 30 minutes with fluorescein isothiocyanate- (FITC)conjugated donkey anti-goat antibodies (1:40; JacksonImmunoResearch, West Grove, PA). The sections werethen rinsed again and mounted with a mixture of glycerol/PBS (3:1) containing 0.1% p-phenylenediamine (PPD) toretard fading (Johnson and de C Nougueira Araujo, 1981;Platt and Michael, 1983), examined, and photographed inan Olympus BX-60 or the Nikon Microphot-FX microscopeequipped with epifluorescence and proper filter combina-tions for FITC.

The sections processed for immunoperoxidase histochem-istry were preincubated with BSA-TX for 1 hour, incubatedwith the goat anti-CTB antiserum (1:1,000; List) over-night, rinsed in PBS, incubated with biotinylated horseanti-goat IgG (1:200; Vector ABC kit, Vector, Burlingame,CA) for 30 minutes. The sections were then incubated withavidin-biotin-peroxidase complex (1:100; Vector), followedby a medium containing 70 mg of 3,38-diaminobenzidinetetrahydrochloride, 200 mg of glucose, 40 mg of ammo-nium chloride, 3 g of nickel ammonium sulfate, and 1 mg ofglucose oxidase (Sigma, St. Louis, MO) in 100 ml 0.1 Macetate buffer (Hsu et al., 1981). Thereafter, they weredehydrated in a graded series of ethanol, cleared in xylene,mounted with Entellan (Merck), coverslipped, and exam-ined and photographed in the Nikon microscope underbrightfield illumination.

B4 binding and CTB immunohistochemistry

Cryostat sections of rat and monkey spinal cord andDRG were preincubated with BSA-TX for 1 hour, andincubated with isolectin B4 from Griffonia simplicifolia I(B4; 10 µg/ml, diluted in BSA-TX; Vector) at room tempera-ture overnight. After rinsing in PBS (3 3 10 minutes), thesections were incubated with a mixture of goat anti-Griffonia simplicifolia I antiserum (1:1,000; Vector) andrabbit anti-CTB (1:500; List) for 18–24 hours at 4°C. Afterrinsing in PBS, the sections were incubated with a mixtureof FITC-conjugated donkey anti-goat IgG (1:40; Jackson)and Lissamine rhodamine (LRSC)-conjugated donkey anti-rabbit IgG (1:40; Jackson) for 30 minutes, rinsed in PBSand mounted with PPD, and examined and photographedin the Olympus or Nikon microscope equipped with epifluo-rescence and proper filter combinations for FITC andLRSC.

Control for immunohistochemistry

Control experiments were conducted by preabsorption ofantisera with CTB (List) or B4 (Sigma), respectively, at theconcentrations of 100 µg/ml and 10 µg/ml for 24 hours at4°C.

Quantification

The DRG sections used for quantification were pro-cessed for immunoperoxidase/HRP histochemistry andwere subsequently slightly counterstained by toluidineblue. They were then examined under brightfield illumina-tion in the microscope using a 203 objective lens. OnlyDRG neuron profiles with a clear nucleus were included inthe study.

To determine the percentage of CTB labelled neuronprofiles, counting was made in all L5 DRGs in both ratsand monkeys except for the left L5 DRGs of the controlrats. Every 15th serial section of the rat DRG (five sectionsper ganglion) and every 80th serial sections of the monkeyDRG (three sections per ganglion) were selected for count-ing. Both labelled and unlabelled neuron profiles werecounted. A total number of 814–1,086 neuron profiles fromeach rat DRG and 1,114–1,417 neuron profiles from eachmonkey DRG were counted. All data were processed foranalysis of variance and the unpaired two-tailed t-test.Results were presented as mean 6 standard error of mean(SEM).

To determine the size of CTB immunoreactive DRGneuron profiles, the square area was measured ipsilater-ally and contralaterally in axotomized rats and monkeys.Three sections at 280-µm intervals from each rat DRG andtwo sections at 1,400-µm intervals from each monkey DRGwere measured. Both labelled and unlabelled neuronalprofiles were measured. A Leica Q570 image systemconnected to a microscope was used to collect and analysethe data.

In L5 DRGs of rats injected with CTB, a total number of1,903 neuronal profiles (598, 647, 658 for the individualrats, respectively) from the sections of the ipsilateralDRGs and 1,806 (590, 605, 611) from contralateral DRGswere analysed 18 days after axotomy, and 1,785 (581, 595,609) from the ipsilateral DRGs, and 1,890 (620, 634, 636)from contralateral DRGs 126 days after axotomy. In L5DRGs of rats injected with CTB-HRP, a total of 1,780neuronal profiles (587, 593, 600) from the sections ofipsilateral DRGs and 1,875 (611, 627, 637) from the

PLASTICITY OF UPTAKE AND TRANSGANGLIONIC TRANSPORT OF CTB 145

contralateral DRGs were measured 18 days after axotomy.In monkey L5 DRGs, a total of 2,945 neuronal profiles(963, 970, 1,012) from ipsilateral DRGs and 3,170 (1,013,1,031, 1,126) from the contralateral DRGs were measured20 days after axotomy, and a total of 2,958 (955, 972, 1,031)from the ipsilateral DRGs and 3,343 (998, 1,109, 1,236)from the contralateral DRGs were measured 96 days afteraxotomy. A Leica Q570 image system equipped with amicroscope was used to collect and analyse the data. Thedata were presented as total number of neuron profilesand labelled ones in the ipsilateral and contralateral L5DRGs of three animals at each time interval vs. neuronprofiles cross-sectional area indexes with 200-µm2 inter-vals.

RESULTS

CTB-labelled neurons in monkey DRGs

In the L5 DRGs of unilaterally axotomized monkeys, thesize measurements of all neuron profiles revealed a major-ity of small neuron profiles, among which more than 80%had a cross-sectional area of 1,200 µm2 or less (Fig. 1a).The distribution profile was very similar for ipsilateral andcontralateral ganglia (Fig. 1a). Similar results were ob-tained 96 days after SNT (Fig. 1c). The number of CTB-labelled neuron profiles was markedly increased in ipsilat-eral L5 DRGs, 20 days after SNT, as compared with thecontralateral side, from 11.5 6 1.5% up to 73.1 6 4.1% ofthe counted total number of neuron profiles (P , 0.01,

Fig. 1. a–d: Histograms showing size (cross-sectional area) ofneuron profiles in general (a,c), of cholera toxin B-subunit– (CTB)labelled profiles vs. all profiles (b,d) in ipsilateral and contralateral L5dorsal root ganglia (DRGs) of monkey 20 (a,b) and 96 (c,d) days (threemonkeys at each time interval) after unilateral sciatic nerve transsec-

tion. a,c: The size distribution of neuron profiles is similar in the DRGson the two sides both 20 and 96 days after axotomy. b,d: In ipsilateralDRGs, the peak of CTB-labelled neurons is shifted to small neuronprofiles (peak at 400–800 µm2 ipsilaterally vs. 800–1400 µm2 contralat-erally).


ipsilateral vs. contralateral DRGs) (Fig. 2a,b). The CTBlabelling was observed in neuron profiles of different sizeswith a peak at 400–800 µm2, showing that they weremainly small (Fig. 1b). In contrast, the contralateralCTB-labelled neuron profiles were larger with a peak at800–1,400 µm2 (Fig. 1b). The increase in the number ofCTB-labelled neuron profiles was also seen in ipsilateralDRGs 96 days after unilateral SNT (70.4 6 3.9% ofcounted neuron profiles vs. 10.8 6 1.8% on the ipsilateralside) (P , 0.01, as compared with contralateral DRGs).

CTB-labelled fibers in the monkeydorsal horn

The distribution of CTB labelling in the ipsilateral andcontralateral dorsal horn 20 days after unilateral axotomyis shown in Figure 3a–d. In the contralateral dorsal horn,only a moderately dense CTB labelling was observed inlaminae I and II with scattered CTB-positive nerve fibersin laminae III and IV (Fig. 3b,d). CTB labelling was notdetected in laminae V and VI, but many motoneurons inboth the ipsilateral and contralateral ventral horns werelabelled by CTB (not shown). Twenty days after axotomy,the number of labelled fibers was markedly increased inlaminae I and II, where distinct patches of densely packedfibers in inner lamina II presented a special feature(Fig. 3a,c). There was a small increase in the number ofCTB-labelled nerve fibers in laminae III and IV (Fig. 3a).These changes were also observed in the ipsilateral dorsalhorn 96 days after sciatic nerve cut (not shown).

B4 binding sites in monkey DRGs

The distribution of B4 binding neuron profiles in ipsilat-eral and contralateral DRGs 20 days after unilateral SNTis shown in Figure 2c,d. B4 binding sites were seen inmany small neuron profiles (60.3 6 2.9% of countedprofiles) in contralateral DRG (Fig. 2d). The cross-sectional area of the B4 binding-positive neuron profileswas in the range of 400–2,400 µm2, with a peak at600–1,600 µm2 (Fig. 4a). No large DRG neuron profileswith B4 binding sites were detected. In ipsilateral DRGs,only 0.8 6 0.2% of counted neuron profiles bound B4 inipsilateral L5 DRG 20 days after unilateral SNT (Fig. 2c)(P , 0.01, compared with contralateral DRGs). Thisfinding is in parallel to the marked increase in CTB-positive neuron profiles (Fig. 2a; the same section asshown in 2c after processing for double labelling). Incontralateral ganglia, around 50% of the CTB-positiveneurons also bound B4 as shown after double labelling forCTB and B4 (cf. Fig. 2e with f).

B4 binding sites in the L4 and L5 monkeydorsal horn

A marked difference in B4 binding was observed be-tween the ipsilateral and contralateral dorsal horn(Fig. 3e,f). Thus, intense B4 binding sites were localized inthe contralateral inner lamina II, forming distinct patches(Fig. 3f). These patches were similar in appearance andlocalization to the patches formed by CTB-positive fibersin the ipsilateral inner lamina II (cf. Fig. 3f with a,c).Lower levels of B4 binding sites were present in outerlamina II (Fig. 3f). A very low density B4 binding was seenin lamina III. In the ipsilateral dorsal horn, B4 bindingsites were markedly reduced after SNT (Fig. 3e), that is,where strongly labelled CTB-positive fibers are found, asshown in double-labelling experiments (Fig. 3a; Fig. 3a

and e show the same section). However, a few smallpatches of weak B4 labelling could be detected in lamina II(Fig. 3e).

CTB-labelled neurons in rat DRGs

In control and contralateral L5 DRGs of the rat, 29.1 61.6% of the counted neuron profiles were large (.1,000µm2), and a very similar size distribution was observed foripsilateral profiles (Fig. 5a). The distribution of CTB-positive DRG neurons 18 days after SNT is shown inFigure 6a–d. In the L5 DRGs, 43.2 6 2.1% and 44.6 6 2.7%of the neuron profiles of, respectively, control rat DRGsand contralateral DRGs were labelled by CTB (differencenot significant). They were mainly large neuron profiles(Figs. 5b, 6b,d). The number of neuron profiles labelled byCTB was markedly increased, up to 81.4 6 2.6% of allneuron profiles, in ipsilateral DRGs 18 days after unilat-eral SNT (Fig. 6a,c) (P , 0.01, compared with the contralat-eral and normal DRGs). These CTB-positive profiles werefound both among small and large DRG neuron profiles(Figs. 5b, 6a,c). Thus, there was a shift in size of ipsilateralCTB-labelled neuron profiles toward small ones 18 daysafter unilateral SNT (Fig. 5b; cf. Fig. 6a,c with b,d). Twopeaks for CTB-labelled profiles were found at 200–800 and800–1,600 µm2, respectively (Fig. 5b). In ipsilateral DRGs,these changes could still be seen 126 days after unilateralsciatic nerve cut. Thus, the percentage of CTB-labelledneuron profiles was increased from 43.5 6 4.2% in contra-lateral DRGs to 79.6 6 4.6% in ipsilateral DRGs (P , 0.01,compared with contralateral and normal DRGs), andagain, CTB-positive profiles were often smaller (Fig. 5f).The size distribution of contra- and ipsilateral neuronprofiles was similar also 126 days after axotomy (Fig. 5e).

CTB-HRP-labelled neurons in rat DRGs

By using CTB-HRP as retrograde tracer, changes simi-lar to those with CTB were observed in the ipsilateralDRGs of the rat 18 days after SNT (Figs. 5c,d; 7a–d). Thus,87.7 6 3.8% of the counted neuron profiles were CTB-HRP-positive in ipsilateral DRGs vs. 54.4 6 4.6% in contralat-eral DRGs (P , 0.01). In the ipsilateral L5 DRGs, theincrease occurred among the small neuronal profiles (0–1,200 µm2) (Fig. 5d), but two peaks could not be observed tothe same extent as after CTB labelling (cf. Fig. 5d and b).There was also a shift in the size distribution of CTB-HRP-positive neuron profiles toward small profiles in ipsilateralDRGs; the highest number of CTB-HRP–positive neuronprofiles was found in the 200- to 800-µm2 range and thesecond peak in the 800- to 1,200-µm2 range (Fig. 5d).

CTB- and CTB-HRP–labelled fibersin rat dorsal horn

Clear differences in CTB and CTB-HRP labelling wereobserved between the ipsilateral and contralateral dorsalhorn (Fig. 8a–c). In contralateral spinal dorsal horn and innormal rats, an intensely stained network of CTB-positivefibers was localized in laminae III–V 14 days after unilat-eral sciatic nerve cut (Fig. 8b,c; normal not shown), withonly scattered CTB-positive fibers in lamina II and a weaklabelling in lamina I (Fig. 8b,c). Eighteen days after SNT,most importantly, strongly CTB-positive nerve fibers ap-peared in laminae I and II (Fig. 8a,c). Furthermore, theintensity of the CTB labelling was increased in the ipsilat-eral laminae III, IV and V, and in ipsilateral dorsal roots(Fig. 8c). Similar changes were also demonstrated in the


Fig. 2. a–f: Immunofluorescence micrographs of ipsilateral (Ipsi)(a, c) and contralateral (Con) (b,d–f) monkey L5 dorsal root ganglia(DRGs) 20 days after unilateral sciatic nerve cut, double labelled forcholera toxin B-subunit (CTB) (a,b,e) and B4 binding sites (c,d,f). a andc, b and d, as well as e and f show the same section, respectively. a–d:In the ipsilateral DRG most neurons of all sizes are labelled by CTB (a)

but no B4-binding neurons can be seen (c). In contralateral DRG (b,d),only some small and large neurons are CTB labelled (b), whereas B4binds to many small neurons (d). e,f: CTB and B4 binding sites arecolocalized in some neurons (arrowheads) in the contralateral DRG.Arrows point to neurons containing B4, but not CTB. Scale bars 5 200µm in a (applies to a–d), 100 µm in e (applies to e,f).


Fig. 3. Immunofluorescence (a,b,e,f) and immunoperoxidase (c,d)micrographs of ipsilateral (Ipsi) (a,c,e) and contralateral (Con) (b,d,f)monkey spinal dorsal horn 20 days after unilateral sciatic nervetransection. a: Intense cholera toxin B-subunit (CTB) labelling is seenin laminae I and II of the ipsilateral dorsal horn with a higherintensity and patchy distribution in inner lamina II. Some CTB-positive fibers are located in lamina III. b: Comparatively few CTB-positive nerve fibers are seen in laminae I and II of the contralateral

dorsal horn. c: Densely packed CTB-positive varicosities form patchesin inner lamina II of the ipsilateral dorsal horn. The number oflabelled fibers is markedly increased in lamina II, compared with thecontralateral side (d). e,f: In the ipsilateral dorsal horn B4 binding isalmost completely absent, whereas strongly stained patches are seenin contralateral inner lamina II (f). Arrowheads in e indicate remain-ing, weakly fluorescent patches. Scale bars 5 100 µm in a (applies toa,b,e,f), 50 µm in c (applies to c,d).


ipsilateral dorsal horn by using CTB-HRP (not shown).These changes could still be seen 126 days after SNT (notshown).

CTB- or CTB-HRP–labelled fibers in gracilenucleus of rat and monkey

Many CTB- (Fig. 8d) or CTB-HRP–labelled primaryafferent fibers were observed in the gracile nucleus ofnormal rats and on the contralateral side after unilateralsciatic nerve cut (Fig. 8d). The number and distribution ofCTB- or CTB-HRP–labelled nerve fibers were increased inthe ipsilateral gracile nucleus (Fig. 8d). CTB labelling wasnot detected in the gracile nucleus of monkeys afterperipheral axotomy, neither on the contralateral nor on theipsilateral side (not shown).

B4 binding sites in rat DRGs

The distribution of B4 binding neurons and their relation-ship with CTB labelling in ipsilateral and contralateral ratDRGs 18 days after unilateral SNT is shown in Figures 4band 6c–f. B4 binding sites were seen in many small neuronprofiles (50.4 6 2.7% of counted profiles) in the contralat-eral DRG (Figs. 4b, 6f). The cross-sectional area of the B4binding-positive neuron profiles were in the range of400–1,200 µm2, with a peak at 600–1,000 µm2 (Fig. 4b). Nolarge DRG neuron profiles with B4 binding sites weredetected. In ipsilateral DRGs, 14.5 6 1.9% of countedneuron profiles bound B4 18 days after unilateral SNT(Figs. 4b, 6e) (P , 0.01, compared with contralateralDRGs). This decrease was in parallel to the markedincrease in CTB-positive neurons (Fig. 6c; same section asshown in Fig. 6e after processing for double labelling). Nocolocalization of B4 binding sites with CTB labelling wasseen in ipsilateral DRGs. In contralateral ganglia, approxi-mately 3.5 6 0.4% of the CTB-positive neurons also boundB4, and 2.0 6 0.3% of the B4-bound neurons were labelledby CTB after double labelling for CTB and B4.

B4 binding sites in the L4 and L5 ratdorsal horn

Intense B4 binding sites were localized in lamina II ofthe contralateral dorsal horn of the spinal cord 18 daysafter axotomy, especially in the inner lamina II, formingdistinct patches (not shown). In the ipsilateral dorsal horn,B4 binding sites were almost completely abolished in themedial lamina II of the spinal cord after SNT (not shown),that is, where strongly labelled CTB-positive fibers arefound.

Control for immunohistochemistry

Preabsorption of antisera with an excess of the corre-sponding CTB and B4 abolished all immunostaining pat-terns described above.

DISCUSSION

Differential CTB labelling in rat and monkeyDRGs and spinal cord

CTB and CTB-HRP have been used for many years asanterograde and transganglionic tracers to demonstratethe central terminal fields of primary afferents (Tro-janowski et al., 1981, 1982; Wan et al., 1982a,b; Lindh etal., 1989; Rivero-Melian and Grant, 1990a,b, 1991; La-Motte et al., 1991; Rivero-Melian et al., 1992). It has beenshown that in the rat CTB and CTB-HRP are mainly takenup by the large DRG neurons. The present study showsthat about 10% of neuron profiles in control monkey L5DRGs are labelled by CTB, which is much lower than in ratDRGs (around 40%). In uninjured rat DRGs, CTB mostlylabelled large neurons with a peak between 800 and 1,600µm2, but also some small neurons; in the monkey, thistracer labels mainly small DRG neurons and some large

Fig. 4. Histograms showing size (cross-sectional area) of neuronprofiles of B4-labelled profiles vs. all profiles in ipsilateral andcontralateral L5 dorsal root ganglia (DRGs) of the monkey (a) and therat (b) 20 or 18 days, respectively, after unilateral SNT (three animalseach group). a: B4-labelled profiles have a peak in the range of small

neuron profiles in the contralateral monkey DRGs, but very fewprofiles are B4-labelled ipsilaterally. b: In contralateral rat L5 DRGs,B4-labelled neuron profiles are small in size. In ipsilateral DRGs, noshift in profile size of B4-positive neurons occurs.


Fig. 5. Histograms showing size (cross-sectional area) of neuronprofiles in general (a,c,e), and of cholera toxin B-subunit (CTB)– (b,f)or CTB-horseradish peroxidase (HRP)– (d) labelled profiles vs. allprofiles in sections of ipsilateral and contralateral rat dorsal rootganglia (DRGs) 18 (a–d) or 126 (e,f) days (three rats at each timeinterval) after unilateral SNT. a,c: The size distributions of neuronprofiles are similar in the DRGs on the two sides. One peak represent-ing small neuron profiles is located at 200–800 µm2, and another onefor larger profiles at 800–1,600 µm2. b,d: The peak of CTB- and

CTB-HRP–labelled neuron profiles is shifted toward small profilesipsilaterally 18 days after axotomy. For CTB, two peaks can be seen,whereas the two peaks are less distinct in the CTB-HRP–labelledmaterial. In the contralateral DRGs the peak for CTB- and CTB-HRP–labelled profiles is in the range of medium-sized and large neuronprofiles. e,f: Ipsilateral and contralateral neuron profiles have asimilar size distribution 126 days after SNT (e). The shift of CTB-labelled neuron profiles to small neuron profiles is also seen inipsilateral DRGs 126 days after axotomy (f).

neurons with a peak between 800 and 1400 µm2. A furtherdifference is observed in the dorsal horn. Thus, in ratspinal cord, numerous CTB-positive fibers are located inlaminae III–VI, which is in sharp contrast to the monkey,in which a network of CTB-positive fibers is confinedmainly to laminae I and II, but with only few labelledfibers in laminae III and IV.

Correlation between CTB- and B4 labellingin DRG and spinal cord

In rat lumbar DRGs, B4 binds to a subpopulation ofsmall neurons representing approximately 50% of thecounted neurons (Wang et al., 1994). They are mainlysmall DRG neurons with a cross-sectional area of 150–

Fig. 6. Immunofluorescence micrographs of ipsilateral (Ipsi) (a,c,e)and contralateral (Con) (b,d,f) rat L5 DRGs 18 days after unilateralsciatic nerve cut, single-labelled for cholera toxin B-subunit (CTB)(a,b), or double-labelled for CTB and B4 binding sites c–f. c and e aswell as d and f show the same section, respectively. a,b: The number ofCTB-labelled neurons is markedly increased in the ipsilateral dorsal

root ganglion (DRG) compared with the contralateral DRG. c,e: In theipsilateral DRG most neurons of all sizes are labelled by CTB (c) butonly a few B4-binding neurons can be seen (e). d,f: In the contralateralDRG, many large neurons and some small neurons are CTB labelled(d), whereas B4 binds to many small neurons (f). Scale bars 5 50 µm ina (applies to a,b), 25 µm in c (applies to c–f).


1450 µm2 (Wang et al., 1994). In the present study, B4binding occurs in 50% of the counted neuron profiles in ratDRGs and 60% in monkey DRGs and is mainly found insmall profiles. With regard to the projection territories ofB4-positive DRG neurons, B4 binding sites are localized onprimary afferent fibers in laminae I and II of rat spinalcord, with higher intensity in inner lamina II and a fewfibers in its outer part (Streit et al., 1985, 1986; Silvermanand Kruger, 1988, 1990; Wang et al., 1994; Molander et al.,1996). Here we point to closely packed, patch-like patternsof B4 binding sites in the inner lamina II of monkey, andsimilar patches positive for CTB were also observed in thisspecies. This pattern is strongly reminiscent of primaryafferent fiber patterns in lamina II of rat (Ygge and Grant,1983; Molander and Grant, 1985) and cat (Rethelyi, 1977)and for somatostatin and galanin (after axotomy) in themonkey dorsal horn (Zhang et al., 1993b).

B4 staining in rat is present in almost all fluoride-resistant acid phosphatase (FRAP), in many calcitoningene-related peptide and substance P, and in all somatosta-tin neurons (Wang et al., 1994). The distribution pattern ofB4-positive primary afferent fibers is also very similar toFRAP-positive nerve fibers in the rat dorsal horn (Knyihar-Csillik and Csillik, 1981). Interestingly, SNT both in ratand monkey induces opposite changes in CTB- and B4labelling of DRG neurons. Thus, whereas CTB uptake wasmarkedly increased, there was a very strong reduction in

B4 binding after SNT. In fact, only a few B4 binding siteswere left in monkey DRGs and lamina II of L4 and L5spinal cord after SNT. In rat DRGs 14% of counted neuronprofiles still bound to B4, which may indicate that theseneurons do not project into the sciatic nerve. As shownpreviously (Molander et al., 1996) B4 binding remains inthe rat lateral dorsal horn. In fact, the decrease in B4binding may be a good marker to identify which DRGs andwhich spinal cord segments have been affected by aperipheral injury.

Increased uptake of CTB in DRG neuronsafter peripheral axotomy

It is a striking finding that over 70% of the DRG neuronprofiles in the monkey are labelled by CTB after SNT (vs.10% contralaterally). This marked increase in CTB-labelled neurons is mainly confined to small neuron pro-files with a size range from 200 to 800 µm2. This findingstrongly indicates that the uptake of CTB can be regulatedin a subpopulation of DRG neurons by SNT. This observa-tion on the monkey stimulated us to extend experiments torats. We then could confirm that CTB- and CTB-HRP-labelled neuron profiles constitute approximately 43% ofall neuron profiles in contralateral L5 rat DRGs, mainlylarge profiles, which is in good agreement with previouslyreported data (42%) (Woolf et al., 1995). Interestingly,

Fig. 7. Immunoperoxidase micrographs of cholera toxin B-subunitconjugated to horseradish peroxidase (CTB-HRP) labelling in ipsilat-eral (Ipsi) (a,c) and contralateral (Con) (b,d) rat dorsal root ganglia(DRGs) 18 days after unilateral sciatic nerve transection. a–d: Thenumber of CTB-HRP-labelled neurons is markedly increased in theipsilateral DRG compared with the contralateral DRG. c: In additionto strongly CTB-HRP-positive large neurons (arrowheads), many

small ipsilateral neurons (curved arrows) are strongly labelled byCTB-HRP. d: In the contralateral DRG, large neurons are strongly(arrowheads) or weakly (arrow) labelled by CTB-HRP. A thin arrowpoints to a weakly labelled small neuron, and a curved arrow to aCTB-HRP-negative small neuron. Scale bars 5 100 µm in a (applies toa,b), 50 µm in c (applies to c,d).


however, a marked increase in the number of CTB- andCTB-HRP-labelled neuron profiles was observed in ipsilat-eral DRGs after unilateral peripheral axotomy also in therat. Thus, more than 80% of the counted neuronal profileswere labelled by CTB or CTB-HRP, and the highestnumber of labelled profiles was now found among smallneuron profiles, indicating a shift toward tracer uptakeinto small DRG neurons after axotomy. Woolf et al. (1995)also analysed DRG cell profiles. Although they also foundlabelled small profiles, a significant difference was notfound between controls and axotomized ganglia (Woolf etal., 1995).

The present data offer another example of changes in thecapability of rat DRG neurons to take up and transporttracer after peripheral axotomy. It has previously beenreported that the isolectin B4 binding sites and also B4transport are markedly reduced in the superficial dorsalhorn of rat spinal cord after peripheral nerve injury(Molander et al., 1996). Taken together, these resultsfurther emphasize the remarkable plasticity that DRGneurons exhibit in response to nerve injury. Thus, theexpression of not only peptides, receptors, and enzymes,but apparently also of surface molecules (see below) isdramatically changed by nerve injury.

Change in uptake of tracers vs. sproutingof primary afferent fibers after axotomy

Peripheral nerve injury induces sprouting of myelinatedprimary afferent fibers into lamina II of rat spinal cord, asreported by Woolf and others (Woolf et al., 1992, 1995;Woolf and Doubell, 1994; Bennett et al., 1996; Lekan et al.,1996; Mannion et al., 1996, 1998; Coggeshall et al., 1997;Doubell et al., 1997), and there is evidence for such aprocess also in monkey on the basis of analysis of longitudi-nal sections of the spinal cord (Florence et al., 1993). Infact, strong evidence for such a process has been obtainedin elegant studies by using intra-axonal HRP filling (Woolfet al., 1992; Koerber et al., 1994). This marked change inthe termination of primary afferents has been proposed torepresent an important anatomical basis for neuropathicpain, which is encountered after peripheral nerve injury(Woolf and Doubell, 1994; Woolf, 1997). The present re-sults demonstrate that the number of CTB- and CTB-HRP–labelled DRG neurons is markedly increased after periph-eral axotomy, especially within the subpopulation of smallneurons, suggesting that at least a large number ofCTB-labelled fibers in laminae I and II might be unmyelin-ated or thinly myelinated primary afferent fibers originat-

Fig. 8. Micrographs of cholera toxin B-subunit– (CTB) labelledipsilateral (Ipsi) (a,c) and contralateral (Con) (b,c) dorsal horn of ratspinal cord (a–c) and gracile nuclei (d) 18 days after unilateral sciaticnerve transsection. a,b: Intense CTB labelling is observed in laminae I(arrowhead), II (curved arrow), III (arrow), and deeper laminae of theipsilateral dorsal horn. Only weak CTB labelling is seen in few nervefibers in the contralateral laminae I and II. Strongly labelled fibers are

seen in deeper laminae. c: The intensity of CTB labelling is alsoincreased in ipsilateral laminae III–VI (cf. arrowheads point at laminaIII) and dorsal roots (curved arrows). d: The intensity and area of CTBlabelling is increased in the ipsilateral gracile nucleus compared withthe contralateral side. Scale bars 5 100 µm in a (applies to a,b), 250µm in c (applies to c,d).


ing from these small neurons. The increased intensity ofCTB in fibers in laminae III–VI after axotomy indicates anincreased uptake of CTB in large DRG neurons. The samemay be true also for the gracile nucleus. The questionwhether the increased CTB- and CTB-HRP–labelling inlaminae I and II would be related to the increased expres-sion of CTB- and CTB-HRP–labelling in small DRGscannot be solved without an analysis of the dorsal rootfibers. The present results, therefore, raise the question towhat extent sprouting of thick-myelinated primary affer-ent fibers from laminae III and IV into laminae I and IIoccurs after peripheral axotomy (for references, see above)and whether the increased binding of CTB to small DRGneurons shown in the present study may be of significancefor the sprouting concept. It at least appears that CTB orCTB-HRP are not proper markers to study these events. Infact, it may be asked whether there are any suitablemarkers available to show an anatomical plasticity ofprimary afferents after peripheral nerve injury, becauseperipheral nerve injury causes marked changes in theexpression not only of neurotransmitters and their recep-tors (Hokfelt et al., 1994, 1997) but also of the bindingsite-mediated uptake of tracers shown in the present and aprevious study (Molander et al., 1996).

It has been reported that nerve growth factor (NGF) canprevent the large primary afferent fiber sprouting intolamina II of the spinal cord after axotomy (Bennett et al.,1996; Eriksson et al., 1997). NGF could possibly haveinfluenced the size distribution of DRG cells in normal,axotomized, and axotomized plus NGF-treated animals.This possibility was investigated in one of the studies, andno significant difference in the distributions of cell diam-eters between the three groups was found (Bennett et al.,1996). In the other study, however, the CTB-HRP–labelledDRG neuronal profiles were found to have a larger area onthe NGF-treated side than on the nontreated side (Eriks-son et al., 1997). The possibility that this could be relatedto a phenotypic switch, resulting in small sized neuronswith unmyelinated axons becoming CTB positive, wasdiscussed (Eriksson et al., 1997). This process was consid-ered less likely, however, because earlier findings hadindicated no change in the area of CTB-HRP–labelledprofiles in the DRG after SNT.

Functional implications of increasedGM1 levels in DRG neuronsafter peripheral axotomy

Because CTB binds to cell membrane glycoconjugates,especially to the monoganglioside GM1 (Cuatrecacas, 1973;Holmgren et al., 1973; King and van Heyningen, 1973),immunohistochemistry for CTB has been used to identifyGM1 monoganglioside in neurons. Thus, the increaseduptake of CTB after peripheral nerve injury may be theresult of elevated GM1 levels in rat DRG neurons. Actu-ally, treatment with neuraminidase, which degrades di-and trisialogangliosides into the monoganglioside GM1,increases CTB labelling in DRGs and the superficial dorsalhorn of rats (Rivero-Melian, 1993). It has been reportedthat the concentrations of mono-, di-, and trisialoganglio-sides are increased eightfold in the crushed optic nerve ofthe goldfish and are involved in nerve regeneration (Sbas-chnig-Agler et al., 1984; Sparrow et al., 1984). GM1 is wellknown to have a protective effect on DRG neurons andstimulate neuritogenesis of DRG neurons (Doherty et al.,1985; Doherty and Walsh, 1987; Cannella et al., 1990;

Barletta et al., 1991). It has been shown that aftercomplete SNT at the midthigh level, 70–80% of all L5 DRGneurons are axotomized (Devor et al., 1985; see alsoAldskogius et al., 1988; Himes and Tessler, 1989). Becausemore than 70% of the lumbar DRG neurons are labelled byCTB in both rat and monkey DRGs after peripheralaxotomy, it appears that almost all injured neurons ex-press GM1. This expression may improve conditions forsurvival and the regenerative capacity of DRG neurons.

In addition to a possible neuroprotective role, GM1 hasbeen shown in cultured DRG neurons to interact withopioid receptors and to convert these receptors from aninhibitory mode to an excitatory one (Shen and Crain,1990; Shen et al., 1991; Crain and Shen, 1992a,b, 1996;Milani et al., 1992; Wu et al., 1997a,b). Neurogenic pain,that is, pain caused by peripheral nerve injury, is difficultto treat and insensitive to opioid analgesics (see Tasker etal., 1983; Arner and Meyerson, 1988, 1993; Kupers et al.,1991; Dray et al., 1994). It is known that the mechanism ofinsensitivity may be related to down-regulation of theexpression of µ-opioid receptors in DRG neurons andneurons in spinal dorsal horn after peripheral axotomy(Jessell et al., 1979; Fields et al., 1980; Lombard et al.,1990; Stevens et al., 1991a,b; Besse et al., 1992a,b; Zhanget al., 1998). Up-regulation of cholecystokinin (CCK) andCCK B receptors in DRG neurons may also contribute(Verge et al., 1993; Zhang et al., 1993a). It might be thatGM1 monoganglioside, present in an increased number ofDRG neurons after peripheral axotomy, also may be in-volved in the interaction with the remaining opioid recep-tors to convert their inhibitory mode to an excitatory one.

Some methodologic aspects

In the present study, we have used the method of profilecounts for quantification only providing estimates of per-centages of labelled vs. total neurons. Thus, we have notattempted to calculate total number of neurons. Thismethod has recently been discussed, and it has beenemphasized that unbiased stereologic methods (Gun-dersen and Jensen, 1987) are preferable (Coggeshall andLekan, 1996). However, it has also been pointed out that,in situations when only rough percentages of a cell popula-tion are required, stereologic methods may not be neces-sary (Saper, 1996). In fact, there is more recent evidencethat the traditional counting method produces more accu-rate counts of neuron in DRGs than did a stereologicapproach (Popken and Farel, 1997; see commentary byGuillery and Herrup, 1997). Considering the very robusteffect (from 10 to 70% of neuron profiles) seen in theanalysis of monkey DRGs, we believe that our quantitativemethod should be adequate. Moreover, in the initial part ofthe study on monkey, the axonal transection was made at arelatively long distance from the cell bodies (at least 200mm), suggesting that cell death should only play a minorrole at least at the shorter survival time (20 days). Withregard to the subsequent analysis in the rat, made forcomparison with the monkey results, we cannot excludethat a certain cell loss and shrinkage could influence themagnitude of changes but not the principal importantconclusions.

The present measurements show that there is a differ-ence between the cross-sectional area of the neuronalprofiles from the animals fixed with a high concentration ofglutaraldehyde (necessary for the CTB-HRP analysis) andthe animals fixed with paraformaldehyde (necessary for


the CTB analysis). Thus, the separation of peaks in therange of small neuronal profiles and large ones is not asdistinct in glutaraldehyde-fixed tissue as in paraformalde-hyde fixed tissue. The more pronounced increase in num-ber of small neuronal profiles in the glutaraldehyde fixedtissue (in the range of 0–1,200 µm2) might result from thefact that glutaraldehyde causes more severe shrinkage ofthe tissue than paraformaldehyde does. In addition, dehy-dration in ethanol and xylene should further enhance theshrinkage. Although there is a similar shift for CTB-HRP–labelled neuronal profiles toward small neuronal profilesas for CTB-labelled ones, the separation of the peaks ofcross-sectional area in small and large neuronal profiles is,again, not as distinct as for CTB-labelled neuronal profiles.It is likely that immunohistochemistry is a better tech-nique to demonstrate the size of CTB-labelled neuronalprofiles.

CONCLUSIONS

In the present study, it was found that peripheralaxotomy causes a marked increase in the uptake andtransganglionic transport of CTB and CTB-HRP, preferen-tially in a subpopulation of small DRG neurons, whichunder normal circumstances does not appear to take upthese tracers, at least to detectable levels. In rat, thischange might lead to an increased number of CTB-labelledprimary afferent fibers in the whole spinal dorsal horn, butespecially of unmyelinated and fine myelinated primaryafferent fibers in laminae I and II and of large myelinatedfibers, unmyelinated fibers, or both, in the gracile nucleus.An even more pronounced increase in uptake of CTBoccurred in monkey DRG neurons after peripheral axotomy.In the monkey spinal cord, however, most CTB-labelledprimary afferent fibers were localized in laminae I and II,and some in lamina III, both normally and after peripheralaxotomy, suggesting differential projection territories ofprimary afferents that take up and transport CTB in ratand monkey spinal cord. The central terminals of unmyelin-ated and fine myelinated afferent fibers, thus, may contrib-ute to the marked increase in CTB-positive fibers inlaminae I and II after axotomy in both rat and monkey.These results show that uptake and transport of CTB canbe regulated in DRG neurons after peripheral axotomy,which may be attributable to the regulation of its bindingsite, GM1 monoganglioside. CTB may thus be a goodtracer or marker for most axotomised DRG neurons ratherthan a selective tracer for large DRG neurons after periph-eral axotomy. Finally, the present results raise the ques-tion on the nature of the dense CTB-positive fiber plexus inthe superficial dorsal horn layers after axotomy, that is therelative contribution of increased CTB uptake in smallDRG neurons vs. sprouting of large neurons from deeperlaminae.

ACKNOWLEDGEMENTS

We thank Ying-Hua Lin, Zhun-Ling Zu, and Xi-Ying Jiaofor their assistance with the surgical procedures andquantification.

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increased uptake and transport of cholera toxin b-subunit in dorsal root ganglion neurons after...

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