Brain Research 863 (2000) 276–281www.elsevier.com/ locate /bres

Short communication

Osteopontin-immunoreactive primary sensory neurons in the rat spinaland trigeminal nervous systemsa,b , c c c*Hiroyuki Ichikawa , Toshiyuki Itota , Yoshihiro Nishitani , Yasuhiro Torii ,

c a,bKiyoshi Inoue , Tomosada SugimotoaSecond Department of Oral Anatomy, Okayama University Dental School, 2-5-1 Shikata-Cho, Okayama 700-8525, Japan

bBiodental Research Center, Okayama University Dental School, 2-5-1 Shikata-cho, Okayama 700-8525, JapancDepartment of Operative Dentistry, Okayama University Dental School, 2-5-1 Shikata-cho, Okayama 700-8525, Japan

Accepted 1 February 2000

Abstract

The distribution of osteopontin-immunoreactive (OPN-ir) primary sensory neurons was investigated in the spinal and trigeminalnervous systems. The dorsal root ganglion (DRG), trigeminal ganglion (TG) and mesencephalic trigeminal tract nucleus (Mes5)contained abundant OPN-ir neuronal cell bodies and their axons. Twenty-five and 31% of neurons were immunoreactive for this protein in

2the DRG and TG, respectively. These neurons were mostly large. A half (54%) of DRG neurons .1200 mm and 9% of those in the2 2range 600–1200 mm showed the immunoreactivity (ir). DRG neurons ,600 mm were mostly devoid of OPN-ir (2%); 31% of TG

2 2neurons exhibited the ir. In the TG, a half (49%) of neurons .800 mm showed the ir and 21% of those in the range 400–800 mm were2immunoreactive for this protein. TG neurons ,400 mm were mostly devoid of OPN-ir (2%). Virtually all (99%) Mes5 primary sensory

neurons exhibited the ir. Muscle spindles in the soleus and masseter muscles contained OPN-ir spiral axon terminals. In the hard palateand incisor periodontal ligament, unencapsulated corpuscular endings exhibited the ir. The co-expression of OPN with parvalbumin andcalcitonin gene-related peptide (CGRP) was also examined in the DRG and TG. In the DRG, virtually all (97%) OPN-ir neuronsexhibited parvalbumin-ir. Conversely, 66% of parvalbumin-ir DRG neurons co-expressed OPN-ir. In the TG, 81% of OPN-ir neuronsexhibited parvalbumin-ir and 69% of parvalbumin-ir ones showed OPN-ir. Virtually all OPN-ir DRG and TG neurons were devoid ofCGRP-ir. The present study indicates that OPN-ir primary sensory neurons in the DRG and Mes5 are spinal and trigeminalproprioceptors. OPN-ir TG neurons appear to include low-threshold mechanoreceptors. 2000 Elsevier Science B.V. All rightsreserved.

Themes: Sensory systems

Topics: Somatic and visceral afferents

Keywords: Calcitonin gene-related peptide; Dorsal root ganglion; Immunohistochemistry; Mesencephalic trigeminal tract nucleus; Osteopontin;Parvalbumin; Trigeminal ganglion

Previous immunohistochemical studies have classified periodontal ligaments, and contain parvalbumin immuno-primary sensory neurons into several subpopulations on the reactivity (-ir) [4]. Thus, this CaBP appears to be a markerbasis of their chemical markers. Parvalbumin, a member of for proprioceptors in the spinal and trigeminal nervouscalcium-binding protein (CaBP) family, is localized to systems. In the TG, however, low-threshold mech-large neurons in the dorsal root (DRG) and trigeminal anoreceptors also contain parvalbumin-ir [6,10]. On theganglia (TG) [4,6,9,10]. Such neurons are considered to be other hand, calcitonin gene-related peptide (CGRP) isproprioceptors in the DRG [4,9]. In the trigeminal nervous considered to be a marker for small to medium-sizedsystem, cell bodies of proprioceptors are located in the neurons in the DRG and TG [11,13,18,19]. CGRP-irmesencephalic trigeminal tract nucleus (Mes5). These primary sensory neurons supply their peripheral receptiveproprioceptors innervate matiscatory muscles and field with free nerve endings [13,18], and are thought to

participate in nociception and/or thermal sensation.*Corresponding author. Fax: 181-86-235-6612. Osteopontin (OPN), a bone matrix protein, has residues

0006-8993/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 00 )02126-0

H. Ichikawa et al. / Brain Research 863 (2000) 276 –281 277

of a calcium-binding amino acid [5,17]. This CaBP has 1. The distribution and cell size of primary sensorybeen shown to be expressed by several tissues including neurons in the DRG, TG and Mes5the bone and kidney [1]. In the nervous system, OPN islocalized to neurons of the vestibular and auditory ganglia The DRG, TG and Mes5 contained abundant OPN-ir[14]. This protein is considered to regulate intracellular neuronal cell bodies and their axons (Fig. 1A–C). In the

21Ca in these neurons. However, little is known about the DRG and TG, ir granules were distributed within thedistribution of OPN-ir primary neurons in the spinal and cytoplasm of these neurons. In the Mes5, strong DABtrigeminal nervous systems. reaction was observed throughout the cytoplasm. Thick

In this study, we examine the distribution of OPN-ir axons of Mes5 neurons were also strongly immunoreactiveprimary sensory neurons in the DRG, TG and Mes5. In for OPN (Fig. 1C). Twenty-five percent (253/1027) ofaddition, the co-expression of OPN with parvalbumin or DRG neurons were immunoreactive for OPN. As shown inCGRP is also investigated in the DRG and TG. Fig. 2, OPN-ir DRG neurons were mostly large and

2 2Six DRGs of the fourth and fifth lumbar segments, four measured 447–4398 mm (mean6S.D.521176777 mm ).2TGs, four brainstems containing the Mes5, soleus and Fifty-four percent (216/399) of DRG neurons .1200 mm2masseter muscles, hard palates and mandibles were ob- and 9% (33/364) of those in the range 600–1200 mm

2tained from six male Sprague–Dawley rats (200–300 g). showed the ir. DRG neurons ,600 mm were mostlyRats were anesthetized with ether to the level at which devoid of OPN-ir (2% or 4/264). Thirty-one percent (305/respiration was markedly suppressed, and transvascularly 997) of TG neurons exhibited the ir. As shown in Fig. 3,perfused with 50 ml of saline followed by 500 ml of 4% OPN-ir TG neurons were mostly large (range5349–4040

2 2formaldehyde in 0.1 M phosphate buffer (pH 7.4). The mm , mean6S.D.512926681 mm ). A half (49% or 232/2materials were dissected and post-fixed with the same 474) of TG neurons .800 mm showed the ir and 21%

2fixative for 30 min. Mandibles were decalcified with (69/325) of those in the range 400–800 mm were24.13% ethylenediaminetetraacetic acid disodium salt in 0.1 immunoreactive for this protein. TG neurons ,400 mm

M phosphate buffer (pH 7.4) for 1 week at room tempera- were mostly devoid of OPN-ir (2% or 4/198). Virtually allture. All materials were soaked in a phosphate-buffered (364/365) of Mes5 primary sensory neurons exhibited the20% sucrose solution overnight, frozen sectioned at 12 ir.mm, and thaw-mounted on gelatin-coated glass slides. Forthe demonstration of OPN, an ABC (avidin–biotin–horse-radish peroxidase complex) method was performed. Sec-

2. The co-expression of OPN with parvalbumin ortions were incubated with mouse monoclonal anti-rat OPN

CGRP in the DRG and TGantibody (1:20 000, American Research Products) for 24 hat room temperature, followed by biotinylated horse anti-

As described previously [4,9,11,19], the DRG and TGmouse IgG and ABC complex (Vector Laboratories).

contained many parvalbumin- and CGRP-ir neurons. TheseFollowing nickel ammonium sulfate-intensified

neurons were scattered throughout the ganglia. Parval-diaminobenzidine reaction, the sections were dehydrated in

bumin-ir neurons were mostly large and CGRP-ir onesa graded series of alcohols, cleared in xylene and cover-

were small to medium-sized. Parvalbumin- and CGRP-irslipped with Entellan (Merck). For cell size analysis, the

neurons were more abundant than OPN-ir ones in the DRGmicroscopic image (3215) of the cell bodies was projected

and TG. In the DRG, virtually all OPN-ir neurons (97% orover a digitizer tablet using a drawing tube. The cross-

679/702) exhibited parvalbumin-ir (Fig. 1D,E). Converse-sectional area of those cell bodies that contained the

ly, 66% (679/1033) of parvalbumin-ir DRG neurons co-nucleolus was recorded.

expressed OPN-ir. DRG neurons co-expressing OPN andFor simultaneous visualization of OPN with parval-

CGRP could not be detected in this ganglion (0 /582, Fig.bumin or CGRP, a double-immunofluorescence method

1F,G). In the TG, 81% (685/848) of OPN-ir neuronswas used. The sections were incubated for 24 h at 48C with

exhibited parvalbumin-ir, and 69% (685/992) of parval-a mixture of mouse monoclonal anti-OPN antibody (1:500)

bumin-ir ones showed OPN-ir (Fig. 1H,I). Virtually alland either rabbit anti-parvalbumin serum (1:1000, SWant)

(332/333) OPN-ir TG neurons were devoid of CGRP-iror rabbit anti-CGRP serum (1:1000, Peninsula Labs). The

(Fig. 1J,K).sections were then treated with a mixture of fluoresceinisothiocyanate-conjugated donkey anti-mouse IgG (1:100,Jackson ImmunoResearch Labs) and lissamine rhodamineB chloride-conjugated donkey anti-rabbit IgG (1:500, 3. Peripheral tissuesJackson ImmunoResearch Labs). For the control, mousemonoclonal anti-OPN antibody was preabsorbed with The soleus muscle contained OPN-ir thick nerve fibersrecombinant mature OPN (20 mg/ml, R&D Systems). No (Fig. 4A). They entered the capsule of muscle spindles,staining was observed in the control. The specificities of and formed spiral axon terminals closely apposed toother antibodies have been described elsewhere [7,20]. intrafusal muscle fibers.

278 H. Ichikawa et al. / Brain Research 863 (2000) 276 –281

Fig. 1. Microphotographs for OPN (A–C,D,F,H,J), parvalbumin (E,I) and CGRP (G,K) in the DRG (A, D–G), TG (B,H–K) and Mes5 (C). Panels D andE, F and G, H and I, and J and K show the same fields of view, respectively. The DRG, TG and Mes5 contained abundant OPN-ir primary sensory neurons(A–C). A double-immunofluorescence method reveals the co-expression of OPN with parvalbumin but not CGRP (D–K). Many OPN-ir neurons showparvalbumin-ir in the DRG (arrows in D, E) and TG (arrows in H, I). CGRP-ir neurons are devoid of OPN-ir in the DRG (arrowheads in F, G) and TG(arrowheads in J, K). Bars550 mm. Panels A, C and D–K are at the same magnification, respectively.

H. Ichikawa et al. / Brain Research 863 (2000) 276 –281 279

each fiber projected few to several terminal twigs at thedistal end (Fig. 4D). The portion of the lingual periodontalligament adjacent to the tooth root and the tissue coveringthe labial portion of the incisor tooth were devoid ofOPN-ir nerve endings. OPN-ir nerve endings could not beobserved in the tooth pulp or gingiva.

In the masseter muscle, OPN-ir thick fibers formedspiral axon terminals on intrafusal muscle fibers (Fig. 4E).

The present study demonstrated that OPN was localizedto primary sensory neurons in the DRG, TG and Mes5.The cell size finding that OPN-ir neurons are mostly largein the DRG suggests that OPN-ir DRG neurons may beprimary proprioceptors. Because Mes5 neurons are consid-ered to be the exclusive source of the trigeminal prop-rioceptors innervating matiscatory muscles and periodontalligaments, OPN-ir appears to be a marker for primaryproprioceptors in the spinal and trigeminal nervous sys-tems. This suggestion is supported by the present findingsthat muscle spindles in the soleus and masseter musclesFig. 2. A histogram showing the cell size spectrum of OPN-ir and

-immunonegative neurons in the DRG. The data were obtained from 1027 contained OPN-ir spiral axon terminals. As in the DRG,DRG neurons. OPN-ir TG neurons were mostly large and small OPN-ir

TG neurons were very rare. However, most OPN-irUnencapsulated corpuscular endings exhibited OPN-ir in neurons are unlikely to be proprioceptors because the

the hard palate and mandible. In the hard palate, OPN-ir number of proprioceptors is small in the TG [15].nerve fibers ran toward the palatal ruga. At the top of The present double-immunofluorescence study revealedpalatal rugae, these nerve fibers projected a few terminals the co-expression of OPN with parvalbumin but not CGRPtwigs and showed a bush-like appearance (Fig. 4B). OPN- in the DRG and TG. In the DRG, virtually all OPN-irir nerve endings could be observed in the periodontal primary sensory neurons co-expressed parvalbumin-ir.ligament of incisor teeth but not molar teeth. In the part of This finding also supports our suggestion that OPN is athe lingual periodontal ligament of the incisor adjacent to marker for muscular proprioceptors in the DRG, becausethe alveolar bone, OPN-ir nerve fibers left the nerve muscle spindles contain parvalbumin-ir axon terminals [4].bundles and ran towards the tooth root (Fig. 4C). In the Unlike in the DRG, a substantial proportion (20%) ofligament, halfway between the bone and tooth surfaces, OPN-ir neurons were devoid of parvalbumin-ir in the TG.

The smaller population of primary neurons co-expressingOPN and parvalbumin in the TG than in the DRG mayreflect a smaller proportion of proprioceptors in the TG.On the other hand, corpuscular endings in intraoral struc-tures contain parvalbumin [6]. Our previous study indi-cated that these endings originate from parvalbumin-ir TGneurons [6]. Thus, the co-expression of OPN and parval-bumin indicates that OPN-ir TG neurons include low-threshold mechanoreceptors. This suggestion is probablysupported by the present finding that unencapsulatedcorpuscular endings in the hard palate exhibited the ir.

OPN-ir could be observed in unencapsulated corpuscularendings within the incisor periodontal ligament. Themorphology and distribution of these nerve endings resem-bled those of Ruffini-like endings which have been de-scribed previously [8,10,12,16]. Periodontal Ruffini-likeendings are low-threshold mechanoreceptors and prop-rioceptors which originate from the TG and Mes5, respec-tively [2,3]. They are thought to be activated by touch,pressure and movement of teeth during chewing, swallow-ing and speech. Because both TG and Mes5 neuronsFig. 3. A histogram showing the cell size spectrum of OPN-ir andcontain OPN-ir, it remains unclear whether OPN-ir cor--immunonegative neurons in the TG. The data were obtained from 997

TG neurons. puscular endings are derived from the TG or Mes5. In any

280 H. Ichikawa et al. / Brain Research 863 (2000) 276 –281

Fig. 4. Microphotographs for OPN in the soleus muscle (A), hard palate (B), incisor periodontal ligament (C,D) and masseter muscle (E). A musclespindle in the soleus muscle contains OPN-ir spiral axon terminals (arrows in A). An OPN-ir nerve fiber run toward the epithelium of the palatal ruga(arrowheads in B), and projected a few terminal twigs at the top of the ruga (arrows in B). OPN-ir nerve fibers are abundant in nerve bundles (arrowheadsin C) within the periodontal space. These nerve fibers leave the nerve bundle, and project few to several terminal twigs at the distal end (arrows in C andD). Spiral axon terminals in a masseter muscle spindle show OPN-ir (arrows in E). Bars520 mm (A,B,D,E) and 100 mm (C).

case, these endings are probably low-threshold mech- all Mes5 neurons exhibited OPN-ir. Ninety-seven and 80%anorecptors and/or proprioceptors in the periodontal liga- of OPN-ir neurons co-expressed parvalbumin-ir in thement. DRG and TG, respectively. OPN-ir DRG and Mes5

In conclusion, we have described OPN-ir primary neurons appear to be proprioceptors in the spinal andsensory neurons in the DRG, TG and Mes5. OPN-ir trigeminal nervous systems. OPN-ir TG neurons probablyneurons were mostly large in the DRG and TG. Virtually include low-threshold mechanoreceptors.

H. Ichikawa et al. / Brain Research 863 (2000) 276 –281 281

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