Horseradish peroxidase conjugated with wheat germ agglutinin (HRP-WGA) was injected into the muscle branch of the mylohyoid nerve in rats. HRP-labeled neuronal cell bodies were observed ipsilaterally in the caudal portion of the trigeminal mesencephalic nucleus and the ventromedial division of the trigeminal motor nucleus (Vmo.vm). Electron microscope observations were carried out on sections through Vmo.vm containing HRP-labeled cell bodies. Synaptic contacts were found between HRP-labeled axon terminals and HRP-labeled nerve cells. The results suggested that mylohyoid muscle spindle afferents are synaptic contacts on the mylohyoid motoneurons

FURUSAWA KIYOFUMI

NAKAYAMA YOKO

OKUDA DAIZO

TANAKA MIKIKO

TANAKA MIZUHO

YAMAOKA MINORU

YASUDA KOUICHI

application/pdfHorseradish peroxidase conjugated with wheat germ agglutinin (HRP-WGA) was injected into the muscle branch of the mylohyoid nerve in rats. HRP-labeled neuronal cell bodies were observed ipsilaterally in the caudal portion of the trigeminal mesencephalic nucleus and the ventromedial division of the trigeminal motor nucleus (Vmo.vm). Electron microscope observations were carried out on sections through Vmo.vm containing HRP-labeled cell bodies. Synaptic contacts were found between HRP-labeled axon terminals and HRP-labeled nerve cells. The results suggested that mylohyoid muscle spindle afferents are synaptic contacts on the mylohyoid motoneurons.journal articl

YASUDA, KOUICHI

FURUSAWA, KIYOFUMI

NAKAYAMA, YOKO

TANAKA, MIKIKO

OKUDA, DAIZO

TANAKA, MIZUHO

YAMAOKA, MINORU

Matsumoto Dental University Repository

(Original) iA"J?Isctwil}2 26 : 31--3s, 2ooo            key words : Trigeminal nerve - Synapses - Electron microseopy - Mylohyoid muscle m                     TrigeminalmotornucleusSynaptic Connections of Mylohyoid Primary Afferent Terminals Made on Jaw-Opening Muscle Motoneurons in the RatKOUICHI YASUDA KIYOFUMI FURUSAWA YOKO NAKAYAMA MIKIKO TANAKADAIZO OKUDA MIZUHO TANAKA and MINORU YAMAOKAOral and Ma tillofacial Surgei y, Matsumoto Dental University School ofDentistr y                      (Chief: Prof M. Yamaoka)Summary  Horseradish peroxidase conjugated with wheat germ agglutinin (HRP-WGA) was in-jected into the muscle branch of the mylohyoid nerve in rats. HRP-labeled neuronal cellbodies were observed ipsilaterally in the caudal portion ofthe trigeminal mesencephalic nu-cleus and the ventromedial division of the trigeminal motor nucleus (Vmo.vm). Electron mi-croscope observations were carried out on sections through Vmo.vm containing HRP-la-beled cell bodies. Synaptic contacts were found between HRP-labeled axon terminals andHRP-labeled nerve cells. The results suggested that mylohyoid muscle spindle afferentsare synaptic contacts on the mylohyoid motoneurons.  It is well known that the trigeminal mesencephalic nucleus (Vmes) contain primary afferent neu-rons innervating the periodontal ligament receptors and muscle spindles of the jaw-closing musclesand the both primary afferents made synaptic contacts with jaw-closing motoneurons. Although ithas been reported the cat jaw-opening muscles, including the mylohyoid muscle, were formerly be-Iieved to be devoid ofmuscle spindles6', in resent studies`"6' the presence ofmuscle spindles in the ratmylohyoid muscle has been demonstrated electrophysiologically and histologically. In these studies,a few neurons in the Vmes were labeled after injection ofhorseradish peroxidase (HRP) into the my-lohyoid muscle or the mylohyoid muscle branch of the mylohyoid nerve in the cat" and rat7"7'. TheseHRP-labeled cells may come from primary afferent fibers innervating the mylohyoid muscle spin-dlei7).  Recent studies have revealed light or electron-microscopic morphological features of the synapticcontacts made between thejaw-closing muscle spindle afferents and their motoneurons2'8''5'.How-ever, there has been no discussion on the existence of synaptic terminals from mylohyoid musclespindle afferents in the ventromedial division of the trigeminal motor nucleus (Vmo.vm ;jaw-open-ing metoneuron pool)'O'. Our experiments attempted to clarify the ultrastructure ofthe synaptic con-tacts made between primary afferents supplying the mylohyoid muscle and their motoneurons.  The experiments were conducted in three male Wistar rats weighing 200 to 250g. They were anes-(submitted February 25, 2000 ; accepted Apri1 19, 2000)32 Yasuda et al. : Sytiapses of mylohyoid primary afferent terminalsthetized wit•h an intraperitoneal injection ofketamine (O.05-1.0mglg) wit-hout tracheal cannulation.Each rat was placed on a table in the supine position, and the body temperature of the animal wasmaintained at 37-38'C using a heating pad. Under a surgical mieroscope, t-he mylohyoid musclebranch ofthe mylohyoid nerve was exposed. The mylohyoid muscle branch was dissected free imme-diately distal to its insertion into the muscle. The proximal cut end of the branch was suctioned intoa glass micropipette (tip diameter ; 100-150pm) fi11ed with 109c HRP conjugated with wheat germagglutinin (HRP-WGA ; Toyobo) in O.3 M KCI and O.05 M Tris buffer at pH7.6, and !eft for 90-120min."'. The HRP-WGA solution was then washed away from the tissue surrounding the nerve, andthe wound was closed. At 48hr after surgery, the animal was perfused through the ascending aortawith heparinized physiological saline (50-100ml), followed by 400-500ml ofa solution of 1.259c glu-taraldehyde and 19c paraformaldehyde in O.1 M phosphate buffer(pH7.2).The brainstem betweenthe superior colliculus and the upper cervical cord was removed. Serial transverse sections 60 ymthick were cut with a Vibratome, collected, and reacted with tetramethylbenzidine (TMB) accordingto the method of Mesulam"i. The wet sections were examined with a light microscope to select sec-tions of the Vmo.vm containing HRP-labeled cells. The best section per rat was treated with a le/cOs,04 solution in O.1 M phosphate buffer (pH6.0) at room temperature for 45 minutes'i' i` , After the os-mication sections were rapidly dehydrated, infiltrated with Epon, and flat-embedded. The embed-ded sections were then viewed with the light microscope, and the Vmo.vm containing labeled cellswas trimmed out and cut on an ultramicrotome. Contrast was provided by treatment with uranylacetate followed by lead citrate. The ultrathin sections were studied with a JEOL JEM-1200EX llelectron microscope.  The three rats in which HRP-WGA was ap-plied to the mylohyoid muscle branch of the my-lohyoid nerve showed evidence of ant-erogradeand retrograde labeling in the brain stem;thelabeled-cell clusters were seen in the ipsilat-eral Vmes (Fig.1) and Vmo.vm. In each of thethree rats, from three to five HRP-labeled cellswere concentrated in an area ef the ipsilateralVmes extending O.80mm to 1.44mm from thecaudal to rostral pole of the nucleus. As the Ia-beled motoneurons were Iocated within theVmo.vm, the leakage ofHRP-WGA tracer fromthe injection site was denied. Ten to twelveHRP-labeled Vmo.vm neurons were observedwithin four or five wet serial sections per rat,and we selected theof a thousand or more ultrathin serial sections./.et,,t- / tve/t,-.Åq,-tu'r.1tt•t-  .v"fi• be,, tg,Fig.1 : Light micrographs ofHRP-labeled cells {ar-     rows) in the Vmes. It was counterstained     with 19c neutral red. Scale bar,100um               best section per rat. The electron microscopic observations were based on study                                         . Twelve synaptic contacts were found between theHRP-labeled terminals and the HRP-labeled motoneurons. The HRP-labeled terminals were foundto make contact with the soma (Fig.2 A, B) and dendrite (Fig.3). Many synaptic contacts were ob-served between the terminals and the motoneurons near the neuronal cell body.  Primary afferent•s arising from the Vmes, which innervate periodontal ligament receptors orjaw-closing muscle spindles" !i' iL", rarely project to thejaw-opening motoneuron pool]S'. It is generally ac-cepted that among trigeminal primary afferents only Vmes neurons innervating muscle spindle and?Atjlgtw:•iliE.Fig.2 : Electron micrograph of an HRP-labeled ter-     minal (asterisk) contacting the HRP-la-     beled soma. B shows a higher-power view of     the rectangular area in A. Arrowhead indi-     cates synaptic contacts. Arrows indicate re-     action products. Scale bars,10pm in A, lpm     in B.26(1) 2000 33Fig.3 : Electron micrographs of HRP-labeled ter-      minal (asterisk) contaeting the HRP-la-      beled dendrite (d). Arrowheads indicate      synaptic contacts. Arrows indicate reaction      products. Scalebar, lpm.periodontal ligament project directly to thetrigeminal motor nucleus. In our observations,synaptic contacts were found between HRP-la-beled terminals and HRP-labeled motoneuronsin Vmo.vm. It thus becomes apparent that my-lohyoid motoneurons receive direct input fromVmes primary afferents that may innervate themylohyoid muscle spindle.  It is generally believed that synapses distrib-uted widely upon the motoneuronal somaticand dendritec surface, including the distal partsofdendritic tree. Our results also demonstrated that axon terminals from presumed mylohyoid mus-ele spindle afferents made synapses upon dendrites and the soma. Recent studies have indicatedthatjaw-closing muscle spindle afferents ofthe cat can be classified into two groups (group Ia andgroup ll )i3''5'. The groups Ia and ll afferents send their collaterals into the jaw-closing motoneuronpool of the trigeminal motor nucleus and the supratrigeminal nucleus whereas the proponion of ter-minal distribution in the supratrigeminal nucleus is higher for group U than group Ia. Futher it hasbeen dernonstrated group ll afferent terminals make synaptic contacts on more distal dendrites ofthe motoneurons than do group Ia afferent terminals. Our recent study suggested that a large num-ber of mylohyoid muscle spindle afferents were group Ia, because the mylohyoid primary afferentsdid not project to the supratrigeminal nucleus'7', The present study also indicated a large number ofsynaptic contacts made between group Ia aflrerents and ct-motoneurons near the neuronal cell body.References1) Alvarado-Mallart MR, Batini C, Buisseret-Delmas C and Corvisier J (1975) Trigeminal repre-   sentations of the masticator and extraocular proprioceptors as revealed by horseradish peroxi-34 Yasuda et al. : Synapses ofmylohyoid primary afferent terminals   dase retrograde transport. Exp Brain Res 23 : 167-79.2) Bae YC, Nakagawa S, Yasuda K, Yabuta NH, Yoshida A, Pil PK, Moritani M, Chen K, Nagase   Y, Takemura M and Shigenaga Y (1996) Electron microscopic observation ofjaw-muscle spin-   dle and periodontal afferent terminals in the trigeminal motor and supratrigeminal nuclei in   the cat.JComp Neurol 374:421-35. '3) Carson KA and Mesulam MM (1982) Electron microscopic demonstration ofneural connections   using horseradish peroxidase : a comparison of the tetramethylbenzidine procedure with seven   other histochemical methods. J. Histochem. Cytochem 30 : 425-35.4) Furusawa K, Yamaoka M, Fujimoto K and Kumai T (1994) Role ofproprioceptor in the mylohy-   oid muscle. Brain Res Bull 35 : 233-6.5) Furusawa K, Yasuda K, Okuda D, Tanaka M and Yamaoka M (1996) Central distribution and   peripheral functional propenies of afferent and efferent components of the superior laryngeal   nerve : morphologieal and electrophysiological studies in the rat. J Comp Neurol 375 : 147-56.6) Gottlieb S, Taylor A and Bosley MA (1984) The distribution of afferent neurons in the mesen-   cephalic nucleus ofthe fifth nerve in the cat. J Comp Neurol 228 : 273-83.7) Jacquin MF, Rhoades RW, Enfiejian HL and Egger MD (1983) Organization and morphelogy ef   masticatory neurons in the rat : a retrograde HRP study. J Comp Neurol 218 : 239-56.8) Luo PF, Wang BR and Li JS (1991) Morphological characteristics and terminating patterns of   masseteric neurons of the mesencephalic trigeminal nucleus in the rat : an intracellular horse-   radish peroxidase Iabeling study. J Comp Neurol 303 : 286-99.9) Mesulam MM and Brushart TM (1979) Transganglionic and anterograde transport ofhorserad-   ish peroxidase across dorsal root ganglia : A tetramethylbenzidine method for tracing central   sensory connections ofmuscles and peripheral nerve. Neuroscience 4 : 1107-17.10) Mizuno N, Konishi A and Sato M (1975) Localization of masticatory motoneurons in the cat and   rat by means of retrograde axonal transport of horseradish peroxidase. J Comp Neurol 164 :   105-16.11) Shigenaga Y, Mitsuhiro Y, Yoshida A, Cao CQ and Tsuru H (1988) Morphology of single mesen-   cephalic trigeminal neurons innervating masseter muscle ofthe cat. Brain Res 445 : 392-9.12) Shigenaga Y, Yoshida A, Mitsuhiro Y, Doe K and Suemune S (1988) Morphology of single mes-   encephalic trigeminal neurons innervating periodontal ligament ofthe cat. Brain Res 448 : 331   -8.13) Shigenaga Y, Mitsuhiro Y, Shiraha Y and Tsuru H (1990) Two types ofjaw-muscle spindle af-   ferents in the cat as demonstrated by intra-axonal staining with HRP. Brain Res 514 : 219-37.14) StUrmer C, Bielenberg K and Spatz WB (1981) Electron microscopical identification of3, 3', 5,   5'-tetramethylbenzidine-reacted horseradish peroxidase after retrograde axoplasmic trans-   port. Neurocsi Lett 23:1-5.15) Yabuta NH, Yasuda K, Nagase Y, Yoshida A, Fukunishi Y and Shigenaga Y (1996) Light micro-   scopic observations of the contaets made between two spindle afferent types and alpha-moto-   neurons in the cat. J Comp Neurol 374 : 436-50.16) Yamaoka M, Furusawa K, Fujimoto K, Iguchi K and Kumai T (1992) Muscle spindles in the my-   lohyoid muscle ofrats. Int J Oral Maxillofac Surg 21 : 309-12.17) Yasuda K, Furusawa K, Tanaka M and Yamaoka M (1995) The distribution ofafferent neurons   in trigeminal mesencephalic nucleus and the central projection ofafferent fibers ofthe mylohy-   oid nerve in the rat. Somatosens Mot Res 12 : 309-15..iAtkJlgtatY4 26(1) 2000 35In!})hen : sT.EaR Etzffim=z-mÅrt-ÅrAhEk,bk?reo•S Å}fxi;Ac:oLN1  't'(iMik-, iSi\fiftsc, pliLLI?\:]r, va4i=-'f'(Ili, ajvaJScis'k, NFIilasiut., Mma i?. (kElrJzlstuJk •MSF) l7 )), F (1)evai!i'Rma e:thNJts'3' 5 Jkpt#Ke: horseradish peroxidase conjugated with wheat germ aggluti-nin (HRP-WGA) e iS JK. L , HRp t..ff-E 'tiX Ni ff ma lpL a) N J{E S k Ot' f'ffk ma tff inth e: D vÅr '( tfi st L t: . )t JLi!FL4 Sfi ffkifi -F" v(f HRP es R'rt Ni fiÅ}X nv geL el =' jl Mi ki FP HX" es& a) Fe M4 zz 2 =' j (. JIili ff LX pt) iA fiR ptJl Pt{I paJJ y'p"'B (Vmo.vm) t: ftJt a•.bb 6 tl.t:. 5 6 e: HRP E.di- l•ft1mp#megeL {?E b V.o... e ;,i.,f}:]rfifit;ltefi -(i•sufi L f: 8 t J5 , HRP t:E.di- :•sk "il.t:em*k3Jii2 Ni-,Z,,mageL2 (1) ;'i J-7'JZLtXft7bfefipt,,,. t5 i•Lk. C] 2•L E, (DS.Sge.k tp , =-jl.JkiliffpiiEM,zzi,9,e:retarwgeLeJfi-9'6.wali"mamaMSit*JtwtS,wt.waiSPN fiS,lj`wh= i-E! )iC:IPi "7"JÅqtrC:JX.h-3-6athS,iP,rp," tz t:.

Synaptic Connections of Mylohyoid Primary Afferent Terminals Made on Jaw-Opening Muscle Motoneurons in the Rat

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顎舌骨筋運動ニューロンと一次求心線維のシナプス接合について

顎舌骨筋運動ニューロンと一次求心線維のシナプス接合について

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