Ž .Brain Research 838 1999 205–209www.elsevier.comrlocaterbres

Short communication

Osteocalcin-immunoreactive primary sensory neurons in the rat spinal andtrigeminal nervous systems

Hiroyuki Ichikawa a,c,) , Toshiyuki Itota b, Yasuhiro Torii b, Kiyoshi Inoue b, Tomosada Sugimoto a,c

a Department of Oral Anatomy II, Okayama UniÕersity Dental School, 2-5-1 Shikata-cho, Okayama 700-8525, Japanb Department of OperatiÕe Dentistry, Okayama UniÕersity Dental School, 2-5-1 Shikata-cho, Okayama 700-8525, Japan

c Biodental Research Center, Okayama UniÕersity Dental School, 2-5-1 Shikata-cho, Okayama 700-8525, Japan

Accepted 1 June 1999

Abstract

Ž .Osteocalcin-immunoreactivity OC-ir was examined in spinal and trigeminal primary sensory neurons of the adult rat. Sixteen percentŽ . Ž .of dorsal root ganglion DRG neurons were immunoreactive ir for this protein. These neurons were mostly large and measured

2 Ž 2. 2594–4583 mm mean"S.D.s2243"748 mm . Thirty-four percent of DRG neurons )1200 mm and 4% of those in the range2 2 Ž .600–1200 mm showed the ir. Virtually all DRG neurons -600 mm were devoid of OC-ir. In the trigeminal ganglion TG , 25% of

Ž 2 2.neurons exhibited the ir. Such neurons were of various sizes ranges156–2825 mm , mean"S.D.s1234"543 mm . Forty-fivepercent of TG neurons )800 mm2 and 6% of those -400 mm2 were immunoreactive for this protein. Twelve percent of TG neurons in

2 Ž .the range 400–800 mm showed the ir. In the mesencephalic trigeminal tract nucleus Mes5 , 63% of primary sensory neurons containedOC-ir. Virtually all OC-ir DRG and Mes5 neurons co-expressed parvalbumin-ir but not CGRP-ir. On the other hand, only 31% of OC-irneurons co-expressed parvalbumin-ir and 10% co-expressed CGRP-ir in the TG. The present study indicates that DRG and Mes5 primarysensory neurons co-expressing OC- and parvalbumin-irs are spinal and trigeminal proprioceptors. OC-ir TG neurons which co-expressparvalbumin- and CGRP-irs appear to include low-threshold mechanoreceptors and nociceptors, respectively. q 1999 Elsevier ScienceB.V. All rights reserved.

Keywords: Calcitonin gene-related peptide; Dorsal root ganglion; Immunohistochemistry; Mesencephalic trigeminal tract nucleus; Osteocalcin; Parvalbu-min; Trigeminal ganglion

Previous immunohistochemical studies have classifiedprimary sensory neurons into several subpopulations on

w xthe basis of their chemical markers 9,14,15,18 . CalcitoninŽ .gene-related peptide CGRP is considered to be a marker

specific to small to medium-sized neurons in the dorsalŽ . Ž . w xroot DRG and trigeminal ganglia TG 14,15,18 .

Ž .CGRP-immunoreactive -ir primary sensory neurons sup-ply their peripheral receptive field with free nerve endings,and are thought to participate in nociception andror ther-

w xmal sensation 15,18 . On the other hand, parvalbumin, aŽ .member of calcium-binding protein CaBP family, is lo-

calized to large neuronal cell bodies in the DRG and TGw x9 . Such neurons are considered to be proprioceptors in

w xthe DRG 4,9 . In the trigeminal nervous system, cellbodies of proprioceptors are located in the mesencephalic

) Corresponding author. Fax: q81-86-235-6639

Ž . w xtrigeminal tract nucleus Mes5 3 . These proprioceptorsinnervate matiscatory muscles and periodontal ligaments,

Ž . w xand contain parvalbumin-immunoreactivity -ir 11 . Thus,this CaBP appears to be a marker for proprioceptors in thespinal and trigeminal nervous systems. In the TG, how-ever, low-threshold mechanoreceptors also contain parval-

w xbumin-ir 10,12,13 .Ž .Osteocalcin OC , an extracellular protein of bone, has

a molecular weight of 5700 and contains three calcium-bi-w xnding amino acid residues 5,6,16 . This CaBP is synthe-

sized by osteoblasts, accumulates in bone, and regulatebone formation. By immunohistochemistry, OC has beenalso shown to be highly expressed by other calcifyingtissues and cells such as the dentine, cementum, odonto-

w xblast and cementoblast 1,2 . However, the presence of OChas never been reported in neurons of the central orperipheral nervous systems.

In this study, we examine the distribution of OC in theDRG, TG and Mes5 to know whether primary sensory

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 01710-2

( )H. Ichikawa et al.rBrain Research 838 1999 205–209206

neurons contain this CaBP. In addition, the co-expressionof OC with parvalbumin or CGRP is also investigated.

Eight DRGs of the fourth and fifth lumbar segments,four TGs and four brainstems including the Mes5 were

Ž .obtained from six male Sprague–Dawley rats 200–300 g .The rats were anesthetized with ether to the level at whichrespiration was markedly suppressed, and transvascularlyperfused with 50 ml of saline followed by 500 ml of 4%

( )H. Ichikawa et al.rBrain Research 838 1999 205–209 207

Ž .formaldehyde in 0.1 M phosphate buffer pH 7.4 . Thematerials were dissected, post-fixed with the same fixativefor 30 min, soaked in a phosphate-buffered 20% sucrosesolution overnight and frozen sectioned at 12 mm. For the

Ždemonstration of OC, an ABC avidin–biotin–horseradish.peroxidase complex method was performed. Sections were

Žincubated with goat anti-rat OC serum 1:2000, Biomedi-.cal Technologies for 24 h at room temperature, followed

by biotinylated rabbit anti-goat IgG and ABC-complexŽ .Vector Laboratories . Following nickel ammonium sul-fate-intensified diaminobenzidine reaction, the sectionswere dehydrated in a graded series of alcohols, cleared in

Ž .xylene and cover-slipped with Entellan Merck . For cellŽ .size analysis, the microscopic image =215 of the cell

bodies was projected over a digitizer tablet using a draw-ing tube. The cross-sectional area of those cell bodies thatcontained the nucleolus was recorded.

For simultaneous visualization of OC with parvalbuminor CGRP, a double-immunofluorescence method was used.The sections were incubated for 24 h at room temperature

Ž .with a mixture of goat anti-OC serum 1:100 and eitherŽmouse monoclonal anti-parvalbumin antibody 1:2000,

. ŽSigma or rabbit anti-CGRP serum 1:1000, Peninsula.Laboratories . The sections were then treated with a mix-

ture of fluorescein isothiocyanate-conjugated donkey anti-Žmouse IgG 1:100, Jackson ImmunoResearch Labs, for

.OC and either lissamine rhodamine B chloride-conjugatedŽdonkey anti-mouse IgG 1:200, Jackson ImmunoResearch.Labs, for parvalbumin or lissamine rhodamine B

Žchloride-conjugated donkey anti-rabbit IgG 1:500, Jack-.son ImmunoResearch Labs, for CGRP . For the control,

Žgoat anti-OC serum was preabsorbed with rat OC 20.mgrml, Biogenesis . No staining was observed in the

control. The specificities of other antibodies have beenw xdescribed elsewhere 7,8 .

The DRG, TG and Mes5 contained abundant OC-irŽ .neuronal cell bodies Fig. 1A–C . The ir granules were

distributed within the cytoplasm of these neurons. In someTG neurons, strong DAB reaction was observed through-out the cytoplasm. Axons of such neurons were also

Ž .immunoreactive for OC Fig. 1B .Dorsal root ganglion, OC-ir cells were distributed

Ž .throughout the DRG. Sixteen percent 106r617 of DRGneurons were immunoreactive for OC. As shown in Fig. 2,OC-ir DRG neurons were mostly large and measured

2 Ž 2 .594–4583 mm mean " S.D.s 2243 " 748 mm .Ž .Thirty-four percent 97r284 of DRG neurons )1200

Fig. 2. A histogram showing the cell size spectrum of OC-ir and-immunonegative neurons in the DRG. The data were obtained from 671DRG neurons.

2 Ž .mm and 4% 8r204 of those in the range 600–1200mm2 showed the ir. Virtually all DRG neurons -600

2 Ž .mm were devoid of OC-ir less than 1% or 1r183 . Asw xdescribed previously 9,14,18 , the DRG contained many

parvalbumin- and CGRP-ir neurons. These neurons werescattered throughout the ganglion. Parvalbumin-ir neuronswere mostly large and CGRP-ir ones were small tomedium-sized. Parvalbumin- and CGRP-ir neurons weremore abundant than OC-ir ones. Virtually all OC-ir neu-

Ž . Žrons 98% or 123r125 exhibited parvalbumin-ir Fig. 1D,. Ž .E . Conversely, 25% 123r500 of parvalbumin-ir neurons

co-expressed OC-ir. DRG neurons co-expressing OC andŽ .CGRP could not be detected in this study Fig. 1F, G .

Trigeminal ganglion, OC-ir cells were distributedthroughout the TG. Twenty-five percent of TG neuronsexhibited the ir. As shown in Fig. 3, OC-ir TG neurons

Ž 2were of various sizes ranges156–2825 mm , mean"2 . Ž .S.D.s1234"543 mm . Forty-five percent 218r487 of2 Ž .TG neurons )800 mm and 6% 16r257 of those -400

mm2 were immunoreactive for this protein. Twelve per-cent of TG neuron in the range 400–800 mm2 showed their. The co-expression of OC with parvalbumin or CGRPcould be observed in the TG. As described previously

Ž . Ž . Ž . Ž . Ž .Fig. 1. Microphotographs for osteocalcin OC A–C, D, F, H, J, L, N , parvalbumin E, I, M and calcitonin gene-related peptide CGRP G, K, O in theŽ . Ž . Ž . Ž . Ž . Ž .dorsal root ganglion DRG A, D–G , trigeminal ganglion TG B, H–K and mesencephalic trigeminal tract nucleus Mes5 C, L–O . Panels D and E,

ŽF and G, H and I, J and K, L and M, and N and O show the same fields of view, respectively. The DRG, TG and Mes5 contain OC-ir neurons arrows in. Ž .A–C . An axon of a strongly-stained TG neuron is also immunoreactive for this protein arrowheads in B . OC-ir primary sensory neurons show

Ž . Ž . Ž .parvalbumin-ir in the DRG arrows in D, E , TG arrows in H, I and Mes5 arrows in L, M . The co-expression of OC and CGRP is also observed in oneŽ . Ž . Ž .TG neuron arrows in J, K but not in DRG neurons F, G or Mes5 neurons N, O . Arrowheads in F–K and N, O indicate OC-ir neurons which are

Ž . Ž . Ž . Ž .devoid of parvalbumin- H, I or CGRP-irs F, G, J, K, N, O . Double arrowheads show parvalbumin-ir D, E and CGRP-ir neurons H–K which lackOC-ir. Barss50 mm. Panels D–O are at the same magnification.

( )H. Ichikawa et al.rBrain Research 838 1999 205–209208

Fig. 3. A histogram showing the cell size spectrum of OC-ir and-immunonegative neurons in the TG. The data were obtained from 1103TG neurons.

w x9,14,18 , parvalbumin- and CGRP-ir neurons were scat-tered throughout the ganglion. Parvalbumin-ir neurons weremostly large, whereas CGRP-ir ones were small tomedium-sized. Parvalbumin- and CGRP-ir neurons weremore numerous than OC-ir ones. Forty-two percentŽ .282r679 of parvalbumin-ir neurons showed OC-ir and

Ž .31% 282r918 of OC-ir ones exhibited parvalbumin-irŽ .Fig. 1H, I . Ten percent of OC-ir neurons co-expressed

Ž .CGRP-ir, whereas 3% 33r1395 of CGRP-ir neuronsŽ .were immunoreactive for OC Fig. 1J, K .

Mesencephalic trigeminal tract nucleus, OC-ir primarysensory neurons were distributed throughout the Mes5.

Ž .Sixty-three percent 195r311 of Mes5 primary sensoryw xneurons exhibited the ir. As described previously 11 , the

Mes5 contained abundant parvalbumin-ir primary sensoryneurons. These neurons were scattered throughout thenucleus. Virtually all OC-ir neurons co-expressed parval-

Ž . Ž .bumin-ir 99% or 232r233 and 69% 232r336 of par-Ž .valbumin-ir neurons showed OC-ir Fig. 1L, M . The

Mes5 was devoid of CGRP-ir primary sensory neurons.Thus, the co-expression of OC and CGRP could not be

Ž .observed in the nucleus Fig. 1N, O .The present study demonstrated that OC is localized to

primary sensory neurons in the DRG, TG and Mes5. Thecell size finding that OC was restricted to large neurons inthe DRG suggests that OC-ir DRG neurons may be propri-oceptors. Because the Mes5 neurons are considered to bethe exclusive source of the trigeminal proprioceptors inner-

w xvating matiscatory muscles and periodontal ligaments 3 ,OC-ir appears to be a marker for primary proprioceptors inthe spinal and trigeminal nervous systems. In the TG,however, most OC-ir neurons are unlikely to be proprio-

ceptors. Although primary neuronal cell bodies innervatingthe stretch receptors innervating the extrinsic eye musclesare located in the ophthalmic division of the TG, their

w xnumber is small 17 . In addition, OC-ir was detected inneurons of the entire cell size range. While the proportionof OC-ir cells were greater for larger TG neurons, thesmallest class of TG neurons also included a substantialOC-ir subpopulation. Thus, it is likely that OC-ir TGneurons have a wide variety of sensory modalities.

The present double-immunofluorescence study revealedthe co-expression of OC with parvalbumin and CGRP. Inthe DRG and Mes5, virtually all OC-ir primary sensoryneurons co-expressed parvalbumin-ir. Because parvalbu-min-containing primary sensory neurons innervate muscle

w xspindles 4 , OC-ir neurons which co-expressed parvalbu-min-ir are probably muscular proprioceptors in the DRGand Mes5. Unlike in the DRG, OC-ir neurons which weredevoid of parvalbumin-ir were abundant in the TG. Inaddition, a substantial proportion of OC-ir TG neuronscontained CGRP, a marker for unmyelinated and finely

w xmyelinated nociceptors 14,15,18,19 . These neurons areprobably devoid of parvalbumin-ir, because the co-expres-sion of parvalbumin and CGRP was very rare in the TGw x9 . OC-ir TG neurons which co-express CGRP-ir appearto be oro-facial nociceptors. On the other hand, encapsu-lated and unencapsulated corpuscular endings in the oral

w xmucosa and hard palate contain parvalbumin 10,12,13 .Our previous study indicated that these endings originated

w xfrom parvalbumin-ir large TG neurons 10,12 . Thus, OC-irTG neurons which co-express parvalbumin-ir probablyinclude low-threshold mechanoreceptors.

In conclusion, we have described OC-ir primary sen-sory neurons in the DRG, TG and Mes5. OC-ir wasrestricted to large DRG neurons. In the TG, however,OC-ir was detected in neurons of the entire cell size range.Virtually all OC-ir primary sensory neurons in the DRGand Mes5 co-expressed parvalbumin-ir. These neurons ap-pear to be proprioceptors in the spinal and trigeminalnervous systems. TG neurons which co-express OC- andparvalbumin-irs may include low-threshold mechanorecep-tors. In addition, a substantial proportion of OC-ir TGneurons co-expressed CGRP-ir but not parvalbumin-ir.These neurons are probably oro-facial nociceptors.

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