The Journal of Neuroscience, August 15, 2001, 21(16):6298–6307 Quantitative Analysis of Synaptic Contacts Made between Functionally Identified Oralis Neurons and Trigeminal Motoneurons in Cats Atsushi Yoshida,1 Hideyuki Fukami,1 Yoshitaka Nagase,1 Kwabena Appenteng,2 Shiho Honma,1 Li-Fen Zhang,1 Yong Chul Bae,3 and Yoshio Shigenaga1 1Department of Oral Anatomy and Neurobiology, Graduate School of Dentistry, Osaka University, Suita, Osaka 565-0871, Japan, 2Department of Physiology, University of Ghana Medical School, Accra 2086, Ghana, and 3Department of Oral Anatomy, Kyungpook University School of Dentistry, Taegu 700-422, Korea A previous study revealed that rostrodorsomedial oralis (Vo.r) with a range of two to seven contacts. In five of the six pairs, neurons synapsing on trigeminal motoneurons use GABA and/or individual or groups of two to three terminals contacted differ- glycine as neurotransmitters. To determine the number and spatial ent dendritic branches of a postsynaptic cell. The Vo.r-dl neu- distribution of contacts, injections of biotinamide and horserad- rons innervated a greater number of counter-stained motoneu- ish peroxidase were made into a Vo.r neuron and an ronal somata than did the Vo.r-vm neurons (216 vs 26). Total -motoneuron in the jaw-closing (JC) and jaw-opening (JO) number of contacts per Vo.r neuron was higher for the Vo.r-dl motor nucleus, respectively, in 39 cats. All Vo.r neurons re- than Vo.r-vm neurons (786 vs 72). The present study demon- sponded to low-threshold mechanical stimulation of the oral strates that axonal branches of Vo.r neurons are divided into tissues. Single Vo.r neurons terminating in the JC nucleus two types with different innervation domains on the postsynap- (Vo.r-dl neurons; n  5) issued, on average, 10 times more tic neuron and that they are highly divergent. The overall effect boutons than Vo.r neurons terminating in the JO nucleus exerted by these neurons is predicted to be much greater within (Vo.r-vm neurons; n  5; 4437 vs 445). The Vo.r-dl neuron–JC the JC than JO motoneuron pool. -motoneuron pairs (n  4) made contacts on either the soma– dendritic compartment or dendrites, and the Vo.r-vm neu- Key words: trigeminal; contact; sensorineuron; motoneuron; ron–JO motoneuron pairs (n  2) made contacts on dendrites, neurobiotin; horseradish peroxidase Previous studies revealed that the rostrodorsomedial part (Vo.r) pressed by systemic administration of strychnine or bicuculline, of the oral nucleus (Vo) contains a large number of interneurons whereas application of both APV and CNQX in the trigeminal projecting to either the jaw-closing motor nucleus (Vmo.dl) or the motor nucleus (Vmo) unmasks monosynaptic IPSP in JO mo- jaw-opening motor nucleus (Vmo.vm) in addition to other brain- toneurons. These results indicate that the Vo.r contains inhibitory stem nuclei (Shigenaga et al., 1988a; Olsson and Westberg, 1991; interneurons acting directly either on JC or JO -motoneurons. Yoshida et al., 1994; Westberg et al., 1995). Furthermore, the Vo.r Further light microscopic (LM) study is essential to determine the is characterized as a region that receives projections mainly from net effects produced by the Vo.r neurons on the motoneurons and primary afferents innervating the oral and perioral structures the spatial distribution patterns of their synaptic sites. (Arvidsson and Gobel, 1981; Shigenaga et al., 1986a,b; Tsuru et The morphological and physiological properties of synaptic al., 1989; Takemura et al., 1991; Moritani et al., 1998) as well as connections made between inhibitory cells and their target projections from jaw-muscle spindle afferents (Luo and Dessem, postsynaptic cells have been analyzed extensively in the neocor- 1995; Luo et al., 1995). tex (Thomson et al., 1996; Tamás et al., 1997) and hippocampus Recently, we provided ultrastructural evidence that Vo.r neu- (Buhl et al., 1994; Miles et al., 1996) by using dual intracellular rons make synaptic contacts on either jaw-closing (JC) or jaw- recordings and labeling. In the spinal cord, Fyffe (1991) examined opening (JO) motoneurons and that all Vo.r premotoneurons that synaptic contacts made by single Renshaw cells on single were examined contain pleomorphic vesicles in their terminals -motoneurons and reported that their synapses are restricted to contacting the motoneuronal somata or dendrites with symmetric dendrites. Recent spinal cord studies have focused on morpho- specializations (Shigenaga et al., 2000). In addition, we found that logical analyses of the numbers and spatial distribution of group Vo.r-induced monosynaptic IPSP in JC -motoneurons is sup- Ia synapses on -motoneurons (Burke et al., 1979; Brown and Fyffe, 1981; Redman and Walmsley, 1983; Lüscher and Clamann, Received Dec. 20, 2000; revised May 21, 2001; accepted May 21, 2001. 1992; Burke and Glenn, 1996) to evaluate conceptual models of This work was supported by a grant-in-aid for scientific research from the Ministry the operation of chemical synaptic junctions ( Rall et al., 1967; of Education, Science and Culture of Japan (11671799) to A.Y. and Y.N. K.A. was Rall, 1977; Redman, 1979). supported by a fellowship from the Japan Society for the Promotion of Science. We are grateful to Dr. Michael Dodson for improving the English of the original In the trigeminal motor system, we offered quantitative mor- manuscript. phological data on single jaw-muscle spindle afferent termina- Correspondence should be addressed to Dr. Yoshio Shigenaga, Department of Oral Anatomy and Neurobiology, Graduate School of Dentistry, Osaka University, 1-8 tions in the Vmo.dl (Shigenaga et al., 1990; Kishimoto et al., Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: sigenaga@dent.osaka-u.ac.jp. 1998) and on their synaptic sites made on single JC Copyright © 2001 Society for Neuroscience 0270-6474/01/216298-10$15.00/0 -motoneurons (Yabuta et al., 1996; Yoshida et al., 1999). Com- Yoshida et al. • Contacts between Sensory Neurons and Motoneurons J. Neurosci., August 15, 2001, 21(16):6298–6307 6299 parisons of these data with those of second-order sensory premo- brainstem was removed then and placed in 25% sucrose in 0.1 M PB, pH toneurons are important for determining the morphological prin- 7.4, at 4°C for 1 week. Transverse serial sections (60 m thickness) were ciple of synaptic distribution patterns governed by different cut on a freezing microtome. Sections were washed in 0.1 M PBS andprocessed with 0.04% 3,3-diaminobenzidine tetrahydrochloride (DAB) afferent inputs and for understanding the neural mechanisms and 0.003% H2O2 in 0.1 M PB, pH 7.4. Then, they were washed in 0.05 M underlying motor coordination and masticatory patterns. Tris-buffered saline (TBS), pH 7.6, and incubated overnight at 4°C in Thus, in this study, we analyzed the numbers and spatial streptavidin conjugated with HRP (1:400; Dako, Glostrup, Denmark) in distribution of synaptic contacts made by single Vo.r neurons on 0.05 M TBS containing 1% Triton X-100. After several rinses with TBS, sections were reacted with 0.02% DAB, 0.003% H2O2, and 0.7% nickelsingle JC and JO motoneurons as well as on counter-stained ammonium sulfate in 0.05 M Tris buffer, pH 7.6, for 15 min. They were motoneurons. washed then in 0.05 M Tris buffer, pH 7.6, and mounted on slides coated with chrome-alum and gelatin. Finally, they were dried overnight, MATERIALS AND METHODS counter-stained with neutral red, dehydrated in graded alcohols, cleared in xylene, and coverslipped. Surg ical preparations. Experiments were conducted on 39 adult cats in Reconstruction of labeled neurons. Motoneurons and axon collaterals of the weight range 2.0–4.2 kg. Anesthesia was initially induced by ket- Vo.r neurons that made synaptic contacts were reconstructed from mul- amine (35 mg/kg, i.m.) followed by sodium pentobarbital (40 mg/kg, i.v.), tiple sections using camera lucida. Objectives of 20 or 50 were used with supplementary doses of sodium pentobarbital (10 mg/ml) being (200 or 500 total magnifications; Olympus Optical, Tokyo, Japan). A given as necessary to maintain a deep level of anesthesia throughout the 100 oil immersion objective was used to reconstruct collateral termi- experiment. End-tidal %CO2, electrocardiogram (ECG), and rectal tem- nations (1340 total magnification) and to identify points of possible perature were monitored continuously and maintained within physiolog- synaptic contact (1000 total magnification). Photomicrographs of the ical limits, and the depth of anesthesia was monitored frequently by contacts were taken by using a 100 oil immersion objective. As was checking the pupil size and pulse rate. All animal procedures were shown in our previous studies (Yabuta et al., 1996; Yoshida et al., 1999), reviewed and approved by the Osaka University Faculty of Dentistry possible contacts were only accepted if all of the following criteria were Intramural Animal Care and Use Committee. satisfied: all stained contacts should be traced back to the stem axon of The preparations were essentially as described in detail by Yoshida et the labeled Vo.r neuron, the contact should consist of a clear terminal al. (1994). In brief, the masseter and mylohyoid nerves were exposed, and bouton or an en passant bouton, and there should be no gap between the bipolar electrodes were placed around them. Bipolar electrodes were also presynaptic and postsynaptic profiles at the same focus (Brown and Fyffe, placed in the mandibular canal for stimulation of the inferior alveolar 1981; Markram et al., 1997). In the present study, Vo.r neurons were nerve and in the infraorbital canal for stimulation of the infraorbital injected with Nb, and trigeminal motoneurons were injected with HRP. nerve. Then, animals were placed in a stereotaxic frame, and after a Premotoneurons and motoneurons were then visualized with and with- craniotomy, parts of the occipital cortex, tentorium, and cerebellum were out nickel enhancement, respectively. This technique tinged collaterals removed to expose the brainstem caudal to the inferior colliculi. Cister- and boutons of the Vo.r neurons and the motoneurons with black and nal drainage and pneumothorax were performed to reduce pulsations of light brown, respectively, making it easy to confirm the presence of the brainstem. Animals were then immobilized with pancuronium bro- contacts between the two elements. mide (0.07 mg/kg, i.v.) and artificially ventilated. Measurements of boutons and somata of motoneurons. Counts of labeled Electrodes and tracers. Intracellular recordings were made using glass boutons and counter-stained motoneurons in the Vmo were made using microelectrodes (borosilicate glass; 1.5 mm outer diameter), filled with a 50 oil-immersion objective (500 total magnification). Counts of the either 5% HRP (Toyobo, Osaka, Japan) or 3% biotinamide [Neurobiotin counter-stained neurons were performed using the optical dissector (Nb); Vector Laboratories, Burlingame, CA], both of which were dis- method (Coggeshall and Lekan, 1996), with counting being restricted to solved in 0.3 M KCl and 0.05 M Tris buffer, pH 7.6. Electrodes filled with the structures with both a clear nucleolus and clearly delineated soma. HRP were used to make recordings from motoneurons, whereas Nb- Small counter-stained neurons with the diameter being 20 m, which filled electrodes were used to record from neurons in the Vo.r. In either had piriformis-like or spindle-like soma with less number of primary case, the electrodes were beveled to resistances of 15–30 M. dendrites, were not counted, because those neurons displayed a feature Recording and labeling procedures. We first located the Vmo on the common to intranuclear neurons (Shigenaga et al., 1988b). The soma basis of the antidromic field potentials that were recorded in response to diameter was measured on a drawing reconstructed in a transverse plane electrical stimulation of the muscle nerves (single pulse with 0.2 msec using the 50 oil-immersion lens (500 total magnification). Measure- duration at 1 Hz) and then identified masseter (JC) and JO motoneurons ments of bouton diameters were made from camera lucida drawings on the basis of the antidromic spike potentials recorded intracellularly. using the 100 oil-immersion lens (1340 total magnification). These Injection of HRP into motoneurons (resting potential more negative drawings were scanned (at 400 dots per inch) and analyzed using NIH than 50 mV) was made by using positive DC currents of 15–20 nA Image (Wayne Rashand, National Institutes of Health, Bethesda, MD). applied for 1.5–2.5 min. We only attempted to fill a single masseter and No corrections for tissue shrinkage resulting from fixation and histolog- a single JO motoneuron in each animal. ical procedures were made in this study. Comparisons of the means of Subsequently, the HRP electrode was withdrawn, and an electrode two groups were made by the Mann–Whitney U test or Student’s t test at filled with Nb was inserted into the Vo.r. The Vo.r was located on the the p  0.05 level. Photomicrographs were processed and labeled using basis of both the stereotaxic coordinates and the monosynaptic field Photoshop 5.0J (Adobe Systems, San Jose, CA), with only the contrast potential (mean latency, 1.2 msec) that was elicited by single electrical being adjusted during processing. Montages were printed on a Fuji stimuli applied to either the inferior alveolar nerve or the infraorbital Pictography 3000 digital photographic printer (Fuji Photo Film, Tokyo, nerve. Intracellular recordings were obtained from Vo.r neurons, with Japan). intracellular penetrations being initially identified by the appearance of Data from one Vo.r neuron (CL2) with terminals in the Vmo.dl and EPSPs with spike potentials after electrical stimulation of either the one Vo.r neuron (OP2) with terminals in the Vmo.vm, which were used inferior alveolar nerve or the infraorbital nerve (single pulse of 0.2 msec for a previous electron microscopic (EM) study (Shigenaga et al., 2000), duration at 1 Hz). Neurons were characterized physiologically by noting were included in the present LM analysis. These two neurons had been the location of the receptive field and determining the response of the labeled by HRP injections, and three sections containing the Vmo had neuron to mechanical stimulation (e.g., tactile stroking of facial skin and been subjected to the EM analysis from each neuron. In these sections, oral mucosa, pressing and tapping teeth, and stretching lower jaw). After the measurements of numbers of motoneurons, labeled boutons, and determining the electrophysiological characteristics of the neuron, Nb contacts on motoneuronal somata were made from polymerized plastic was iontophoresed with 10–15 nA positive DC current for 1–2 min, even sections before ultrathin sectioning. The remaining sections were ana- if the resting membrane potential decreased (less than 40 mV). We only lyzed according to the protocols described above. attempted to fill a single Vo.r neuron per animal. Histochemical procedures. Animals were allowed to survive for 6–12 hr from the time of the last injection, after which they were deeply anes- RESULTS thetized further and perfused through the ascending aorta with 1.5 l of 0.02 M PBS, pH 7.4. Subsequently, animals were perfused with 4 l of In the present study, injections of tracers (Nb or HRP, see fixative solution of 4% paraformaldehyde in 0.1 M phosphate buffer (PB), Materials and Methods) were made into a Vo.r neuron, a masse- pH 7.4, followed by 1 l of the same fixative containing 10% sucrose. The ter motoneuron (JC), and a jaw-opening (JO) motoneuron in 6300 J. Neurosci., August 15, 2001, 21(16):6298–6307 Yoshida et al. • Contacts between Sensory Neurons and Motoneurons Table 1. Quantitative data for single Vo.r neuron terminals and their contacts on counter-stained motoneuronal somata in the JC motor nucleus and the JO motor nucleus Total number of Number of con- Total number Number (percentage) of motoneuronal Number (percentage) of tacts per soma Case number Receptive field of boutons boutons contacted somata somata contacted contacted Vo.r-dl neuronsa CL1 Lower canine and premolars 6123 1202 (19.6) 1572 278 (17.7) 4.3 CL2 Upper premolars 5855 1168 (19.9) 1656 297 (17.9) 3.9 CL3 Upper premolars 2022 362 (17.9) 1541 112 (7.3) 3.2 CL4 Upper teeth 4565 748 (16.4) 1728 220 (12.7) 3.4 CL5 Lower incisors 3620 450 (12.4) 1674 173 (10.3) 2.6 Mean  SEM 4437  754* 786  175* (17.2  1.4%) 1634  34* 216  34* (13.2  2.1%)* 3.5  0.3 Vo.r-vm neuronsb OP1 Lower premolars and molars 556 94 (16.9) 521 34 (6.5) 2.8 OP2 Lower incisors 471 75 (15.9) 485 33 (6.8) 2.3 OP3 Upper and lower lips 341 48 (14.1) 474 21 (4.4) 2.3 Upper and lower premolars and OP4 molars 449 71 (15.8) 498 25 (5.0) 2.8 Upper gingiva between canine OP5 and first premolar 406 72 (17.7) 480 24 (5.0) 3.0 Mean  SEM 445  36* 72  7* (16.1  0.6%) 492  8* 26  3* (5.3  0.6%)* 2.8  0.1 aIndicates Vo.r neurons that issue collaterals terminating in JC motor nucleus. b Indicates Vo.r neurons that issue collaterals terminating in JO motor nucleus. *Significant differences (p  0.05, Mann–Whitney U test) between Vo.r-dl and Vo.r-vm neurons. each of 39 animals. Twenty-two Nb-labeled Vo.r neurons were gingival neuron (OP5) and lip neuron (OP3) both displayed a found after histochemical processing. Thirteen of these neurons transient response to a maintained light pressure applied to the had axon collaterals that terminated in the Vmo.dl of the Vmo receptive field. Note that none of the Vo.r neurons examined (Vo.r-dl neurons) and nine in the Vmo.vm of the Vmo (Vo.r-vm responded to displacement of the lower jaw, suggesting that these neurons). In the remaining 17 neurons, the labeling was too weak neurons received no input from jaw-muscle spindle afferents. to identify their terminals with a light microscope. Of the 13 An example of the electrophysiological and morphological data Vo.r-dl neurons, 10 each contained an Nb-labeled Vo.r-dl neuron, on a Vo.r-dl neuron CL3 is shown in Figure 1A–C. This neuron an HRP-labeled masseter motoneuron, and an HRP-labeled JO fired two spikes, superimposed on EPSP, in response to a single motoneuron. In four of the 10 cases, contacts made between the electrical stimulation (submaximal) of the infraorbital nerve; the Vo.r-dl neurons (CL1, CL3, CL4, and CL5) and the masseter maximal stimulation increased the duration of the EPSP and motoneurons were found, and these four pairs were reconstructed numbers of the action potentials (data not presented). These and analyzed. An additional HPR-labeled Vo.r-dl neuron CL2 firing patterns were applicable to the other Vo.r neurons exam- that was used for a previous EM study (Shigenaga et al., 2000) was ined and to previously reported Vo.r neurons (Yoshida et al., added to the present analysis. Of the nine Vo.r-vm neurons, eight 1994; Shigenaga et al., 2000), but the number of action potentials contained an Nb-labeled Vo.r-vm neuron, an HRP-labeled JO differed from one cell to another (2–9 spikes). This neuron was motoneuron, and an HRP-labeled masseter motoneuron. In two activated in an FA fashion by a gentle tap applied to upper of the eight pairs, contacts were found between the Vo.r-vm premolars. The latencies of EPSPs generated in five Vo.r-dl neurons (OP1 and OP3) and JO motoneurons, and these two neurons and five Vo.r-vm neurons ranged from 1.2 to 1.7 msec pairs were reconstructed and analyzed. An additional two (mean, 1.4 msec) after a single electrical stimulation of the Vo.r-vm neurons (OP4 and OP5) and an HRP-labeled Vo.r-vm peripheral nerve. As was previously reported (Yoshida et al., neuron OP2 used for a previous EM study (Shigenaga et al., 2000) 1994; Shigenaga et al., 2000), Vo.r neurons were arranged in a were also incorporated in the present analysis. topographic fashion with the somata of Vo.r-dl neurons being General physiology and morphology of Vo.r neurons located more dorsally or laterally than those of Vo.r-vm neurons (Fig. 1D,E). In addition, we confirmed our earlier observation All Vo.r-dl neurons (CL1, CL2, CL3, CL4, and CL5) were acti- (Yoshida et al., 1994) that Vo.r neurons issue collaterals that vated when light mechanical stimulation was applied to the teeth. terminate in the brainstem nuclei other than the Vmo [e.g., Four neurons (CL1, CL2, CL3, and CL5) were of the fast adapt- principal nucleus (Vp), Vo, intertrigeminal region, and juxtatri- ing (FA) type, whereas the other neuron (CL4) was a slowly geminal region]. Detailed observation of axonal trajectory in adapting (SA) type. In contrast, all Vo.r-vm neurons (OP1, OP2, regions other than the Vmo and complete reconstructions of OP3, OP4, and OP5) were the FA type. Neurons OP1, OP2, and examined Vo.r neurons except for a Vo.r-dl neuron CL3 (Fig. OP4 were activated by light mechanical stimulation of the teeth 1B,C), however, were not performed. (periodontal ligament), whereas neurons OP3 and OP5 had their receptive field in the lips and gingiva, respectively. The location Contacts made by single Vo.r neurons and of their receptive fields is listed in Table 1. Fast-adapting neurons single motoneurons were characterized by their transient response to a tap of the Contacts on JC motoneurons tooth without directional sensitivity, whereas the SA periodontal The masseter motoneurons were identified by intracellular re- neuron CL4 responded phasically with directional sensitivity to cordings of antidromic responses after stimulation of the masseter gentle deformation in one of the four orthogonal directions. The nerve. Antidromic spike potentials of the four masseter motoneu- Yoshida et al. • Contacts between Sensory Neurons and Motoneurons J. Neurosci., August 15, 2001, 21(16):6298–6307 6301 Figure 1. Physiology and morphology of a la- beled neuron CL3 in the rostrodorsomedial part (Vo.r) of oral nucleus (A–C) and the somal location of labeled Vo.r neurons examined (D, E). A, Eight superimposed traces of intracellu- lar potentials recorded from the Vo.r neuron CL3 after stimulation of the infraorbital nerve with submaximal intensity. B, Camera lucida drawings of soma-dendrites and part of the stem axon (arrowheads) of the Vo.r neuron. C, Diagram of axonal trajectory of the Vo.r neu- ron with terminals in the dorsolateral division (Vmo.dl ) of the trigeminal motor nucleus (Vmo) and in the brainstem nuclei other than the Vmo.dl. Contacts made between the Vo.r neuron and labeled masseter motoneuron are marked with arrowheads labeled by S1, S2, and S3. The collaterals from an ascending and a descending axon are denoted by a and d, re- spectively. X indicates the midline. D, E, Somal location of Vo.r neurons examined is plotted in two sections at the rostral ( D) and caudal ( E) levels of the Vo.r. Circles and squares represent Vo.r neurons that project to the Vmo.dl and to the ventromedial division (Vmo.vm) of the Vmo, respectively. Arabic numerals within the circles and squares indicate sample (neuron) numbers. SO, Superior olive; Vint, intertri- geminal region; Vjux, juxtatrigeminal region; Vp, trigeminal principal nucleus; Vpv, ventro- lateral subnucleus of Vp; Vtr, spinal trigeminal tract; 7N, facial nerve; D–M, dorsal–medial. Calibration: A, 10 mV, 4 msec. Scale bars: B, 2 mm; D, E, 1 mm. rons were evoked at a constant latency of 0.6–0.8 msec (mean, 0.7 made a contact on a caudoventrally directed fourth-order den- msec), which compares to a mean latency of 0.8 msec reported for drite of the masseter motoneuron. The collateral a2 bore two en a sample of 23 intracellularly labeled -masseter motoneurons passant boutons that contacted on a ventrally extended fourth- (Yabuta et al., 1996). In one of the four masseter motoneurons order dendrite (S1) and on a dorsomedially extended second- (initial membrane potential, 62 mV), we observed a depolariz- order dendrite (S2), respectively. The physical distance and loca- ing hump in the membrane potential ( Chandler et al., 1994; tion of the individual contacts are diagrammatically illustrated in Kobayashi et al., 1997) that separated the first from slow after- Figure 2B. hyperpolarization (also see Shigenaga et al., 1988b, 2000). The An example of contacts made by a Vo.r-dl neuron CL4 on the soma diameters of the four masseter motoneurons ranged from soma and proximal dendrites of a masseter (JC) motoneuron is 41.4 to 53.9 m with a mean of 47.7 m, and thus belong to a illustrated in Figure 4. In this figure, parts of the motoneuron and large-sized group of masseter motoneuron (see Fig. 9A). Four of collateral branches that constituted contacts were reconstructed. the 10 pairs of Vo.r-dl neuron–JC motoneuron combinations This Vo.r-dl neuron CL4 had an ascending stem axon only, which established contacts (see Materials and Methods for the criteria gave off two collaterals a1 and a2 terminating in the Vmo.dl, but of the contacts). The number of contacts made by each pair was the contacts were made by boutons given off from the collateral a2 4 (CL1), 3 (CL3), 7 (CL4), and 6 (CL5), respectively, with a mean only. Specifically, two en passant boutons (S4, S5) contacted a of 5 ( 0.9, SEM). One pair (CL4) made contacts on the soma rostromedially directed first-order dendrite (Figs. 2C, 4A), one en and proximal dendrites, but the other pairs contacted only den- passant bouton (S6) contacted a rostrodorsolaterally directed drites. Dendritic contacts were located within 550 m from the first-order dendrite (not illustrated in Fig. 4), and one terminal soma, with the exception of contacts made by one pair (CL5), bouton (S7) contacted a dorsally directed second-order dendrite which occurred more distally (Fig. 2). Note that each of the of the motoneuron (Figs. 2C, 4B). The collateral a2 also gave off labeled boutons never contacted two different postsynaptic two terminal boutons (S1, S2) and one en passant bouton (S3), all profiles. of which formed contacts on the soma of the motoneuron (Figs. Figure 3 shows the superimposed drawings that were recon- 2C, 4A). structed from a Vo.r-dl neuron CL3 and a masseter motoneuron. This premotoneuron issued a stem axon that divided into an Contacts on JO motoneurons ascending and a descending axon (Fig. 1C). The ascending axon The JO motoneurons were identified by intracellular recordings issued two collaterals, a1 and a2, that formed terminal arbors in of antidromic responses after stimulation of the mylohyoid nerve. the Vmo.dl. The collateral a1 bore a terminal bouton (S3) that Antidromic spike potentials of the two JO motoneurons were 6302 J. Neurosci., August 15, 2001, 21(16):6298–6307 Yoshida et al. • Contacts between Sensory Neurons and Motoneurons Figure 3. Camera lucida drawings reconstructed from a labeled masseter motoneuron (blue) and a labeled Vo.r neuron CL3 (red) showing three contacts made between the two. Contacts are marked with arrows labeled with S1, S2, and S3. The size of labeled boutons drawn is exaggerated. Note that the intracellular responses, soma-dendrites and a stem axon, and scheme of the axonal trajectory of the Vo.r neuron are shown in Figure 1A–C, respectively. D–M, Dorsal–medial. Scale bar, 0.4 mm. drites (Fig. 2E,F). Each of the labeled boutons made a single contact on a dendrite. An example of contacts made by a Vo.r-vm neuron OP1 on a JO motoneuron is illustrated in Figures 5 and 6. This pair involved a total of six contacts. Figure 5 shows the superimposed drawings that were reconstructed from the Vo.r-vm neuron OP1 and the JO motoneuron. In Figure 6, parts of the collateral branches and dendrites that formed contacts were reconstructed, but note that two contacts, S1 and S2, are not shown. This Vo.r-vm Figure 2. Diagrammatic summary showing the locations of contacts made by four Vo.r-dl neuron–JC motoneuron pairs ( A–D) and two neuron OP1 had a stem axon that divided into an ascending and a Vo.r-vm neuron–JO motoneuron pairs (E, F ), with terminal boutons descending axon. One collateral u2 (united axon) given off from denoted by open triangles and en passant boutons denoted by open circles. the stem axon arising from the soma formed terminal arbors in the JC and JO motoneurons receiving contacts from single Vo.r neurons are Vmo.vm and branched extensively, especially in the ventral part of marked with CL and OP, respectively. In each pair, contacts marked with Sn are arbitrary. Scale bar, 100 m (refers to the geometric dendritic Vmo.vm. This collateral made six contacts on dendrites of the distance from the soma). The distance was measured from reconstruc- motoneuron, with four of the contacts being from en passant tions in the transverse plane and corrected by using the Pythagorean boutons and two from terminal boutons (Figs. 2E, 5, 6). An en theorem and the section thickness. The a, d, and u indicate collaterals passant bouton (S1) contacted a rostroventrally directed sixth- given off from an ascending fiber, a descending fiber, and a stem axon, order dendrite, whereas another (S2) contacted a rostrally di- respectively. Dendrites longer than 700 m in A–C, E, and F and those longer than 1000 m in D are interrupted with a broken line. Arabic rected first-order dendrite. In addition, an en passant bouton (S3) numerals attached to the end of dendritic lines indicate the longest distance and a terminal bouton (S4) both made contact on the same, (m) of the dendritic tree formed by each primary dendrite. Note that the rostromedially directed, fourth-order dendrite (Figs. 2E, 5, 6A). second dendritic line in D starts 550 m from the soma. The axon The remaining en passant bouton (S5), together with a terminal collateral branching patterns and somata are also shown, but not to scale. For further descriptions, see Results. bouton (S6), contacted another rostroventrally directed fourth- order dendrite (Figs. 2E, 5, 6B) that arose from the same primary dendrite as that for the contacts S3 and S4. evoked at a constant latency of 0.92 and 0.63 msec, respectively (note that a depolarizing hump was not seen). Their somal Contacts made by single Vo.r neurons on counter- diameters were 52.1 and 45.7 m, respectively. Two of the six stained motoneuronal somata pairs of Vo.r-vm neuron–JO motoneuron combinations estab- General morphology lished two and six contacts, respectively. All contacts made by the Axon collateral(s) of the Vo.r neurons gave off boutons that two pairs were located within the proximal two-thirds of den- appeared to form numerous patch-like clusters distributed widely Yoshida et al. • Contacts between Sensory Neurons and Motoneurons J. Neurosci., August 15, 2001, 21(16):6298–6307 6303 Figure 5. Camera lucida drawings reconstructed from a labeled jaw- opening (JO) motoneuron and a labeled Vo.r neuron OP1 showing six contacts made between the two. Contacts are marked with arrows labeled with S1, S2, S3, S4, S5, and S6. The size of labeled boutons drawn is exaggerated. D–M, Dorsal–medial. Scale bar, 0.4 mm. than that of a sample of 228 boutons randomly selected from the two Vo.r-vm neurons (1.9  0.0 m). Quantitative analysis Contacts made by the five Vo.r-dl neurons (CL1, CL2, CL3, CL4, and CL5) and five Vo.r-vm neurons (OP1, OP2, OP3, OP4, and OP5) on counter-stained somata were analyzed (Table 1). The average number of boutons given off from single Vo.r-dl neurons Figure 4. Parts of drawings reconstructed from a labeled Vo.r neuron was 9.7 times higher than that from single Vo.r-vm neurons. The CL4 and a labeled masseter motoneuron showing six contacts (S1–5 and average number of somatic contacts made by the Vo.r-dl neurons S7 ) and photomicrographs of the contacts S1 and S7. A, Contacts made by was 11 times higher than that made by the Vo.r-vm neurons, labeled boutons from the Vo.r neuron on the soma (S1, S2, and S3, arrows) and a primary dendrite (S4 and S5, filled arrowheads) of the which rendered a significantly higher proportion of the number of labeled masseter motoneuron and on a counter-stained motoneuronal somata contacted for the Vo.r-dl neurons than for the Vo.r-vm soma (open arrowheads) in the Vmo.dl. Photomicrograph in the inset neurons. The number of contacts per motoneuronal soma was, on shows the contact S1 (arrow). B, Contacts made by a labeled bouton on a average, 3.5 and 2.8 for the Vo.r-dl and Vo.r-vm neurons, respec- proximal dendrite (S7, filled arrowhead) of the labeled motoneuron and two labeled boutons on the soma of a counter-stained motoneuron (open tively, but this difference was not significant. arrowheads) in the Vmo.dl. Photomicrograph in the inset shows the Because the JC motor nucleus contains - and -motoneurons, contact S7 ( filled arrowhead). A contact S6 is not included in sections used in contrast to the JO one that contains -motoneurons only, the for the reconstructions. Note that the labeled soma and the counter- soma diameters of counter-stained JC and JO motoneurons in stained somata are cut into two pieces, and each contact is seen on the cases CL5 and OP3, respectively, were measured. The soma surface of a smaller piece. D–M, Dorsal–medial. Scale bars: reconstruc- tions A and B, 20 m; insets A and B, 5 m. diameters of JC motoneurons showed an asymmetric distribution that was skewed to the peak value (Fig. 9A), whereas those of JO motoneurons were unimodally distributed (Fig. 9B). Although in either the Vmo.dl (Fig. 7A–C) or Vmo.vm (Fig. 7D–F). Of the somata in the JC motor nucleus were significantly larger than these boutons, significant numbers were found to contact the those in the JO motor nucleus [42.0  0.3 m (n  625) vs 36.0  somata (Fig. 8 ) and/or juxtasomatic regions (Fig. 8 ) of mo- 0.3 m (n  370)], the different distribution patterns indicate thatA B -motoneurons are contained within the smallest group of the JC toneurons stained with neutral red. motoneuron. This suggests that soma sizes of JO -motoneurons The boutons of both Vo.r-dl and Vo.r-vm neurons consisted of and JC -motoneurons partly overlap each other. Judging from an en passant type and a terminal type. A total of 1022 boutons the size distribution of somata contacted (closed columns in Fig. sampled randomly from the three Vo.r-dl (CL1, CL4, and CL5) 9A) in the JC motor nucleus, JC motoneurons involved in the and two Vo.r-vm neurons (OP1, OP3) showed a similar propor- small group receive less frequent contacts from terminals of tion of en passant boutons and terminal boutons: 60% (618/ Vo.r-dl neurons. Note that each of the labeled boutons never 1022) were of en passant type, and measurements of the diame- contacted two different counter-stained somata. ters of 256 boutons showed no difference for boutons of the en passant and terminal type. However, the diameter of a sample of DISCUSSION 908 boutons randomly selected from the three Vo.r-dl neurons Intracellular labeling of single second-order sensory neurons in was 1.7  0.0 m (mean  SEM), which was significantly smaller the Vo.r and single trigeminal motoneurons revealed that two 6304 J. Neurosci., August 15, 2001, 21(16):6298–6307 Yoshida et al. • Contacts between Sensory Neurons and Motoneurons Figure 7. Distribution patterns of labeled boutons from a Vo.r neuron Figure 6. Parts of drawings illustrated in Figure 5 showing the contacts CL1 in the Vmo.dl (Vo.r-dl neuron; A–C) and from a Vo.r neuron OP1 in S3 and S4 (A) and the contacts S5 and S6 (B) at higher magnification and the Vmo.vm (Vo.r-vm neuron; D–F). The Vo.r-dl neuron boutons found photomicrographs of the contacts S3 and S6. A, Contacts made by two in every alternate section at levels of the rostral (A), middle (B), and labeled boutons on the same dendrite of the labeled JO motoneuron (S3 caudal one-third (C) of the Vmo are superimposed in one representative and S4, filled arrowheads). Photomicrograph in the inset shows the contact section, respectively. The Vo.r-vm neuron boutons found in serial sections S3 ( filled arrowhead). B, Contacts made by two labeled boutons on the at the rostral (D), middle (E), and caudal (F) levels of the Vmo are same dendrite of the labeled motoneuron (S5 and S6, filled arrowheads). superimposed in one representative section, respectively. Boutons con- Photomicrograph in the inset shows the S6 contact ( filled arrowhead). tacting the somata and/or juxtasomatic regions of counter-stained mo- Note that labeled boutons make contacts with the soma of two counter- toneurons are marked with large dots. Boutons without contacts on the stained JO motoneurons (open arrowheads). D–M, Dorsal–medial. Scale counter-stained motoneurons are marked with small dots. Dor–Med, Dor- bars: reconstructions A and B, 20 m; insets A and B, 5 m. sal–medial. Scale bar, 0.5 mm. kinds of neuron are connected monosynaptically with a range of 2–7 synaptic sites. The premotoneuron gave off two types of Another problem is that the soma size of counter-stained JC axonal branches: one innervating the soma–dendritic compart- motoneurons did not show a clear bimodal distribution. It is, ment and the other dendrites of the motoneurons. Premotoneu- however, possible to consider that -motoneurons are contained rons innervating JC motoneurons show a stronger degree of in the smallest group of the JC motoneurons. Because the mor- divergence than those innervating JO motoneurons. The func- phologic and physiologic criteria for distinguishing - from tional implications of neural circuits made between trigeminal -motoneurons have not been determined in the trigeminal JC sensory and motor neurons are discussed. motoneurons, we did not take -motoneurons into consideration Technical considerations in the present study. In the present LM observation, one possible limitation is the lack Comparisons of contacts made by premotoneurons on of confirmation that a closed apposition represents a synapse at JC and JO motoneurons the EM level. In this respect, we confirmed that axon varicoses In the present study, two approaches were used to provide direct forming these close appositions are synapses in a previous EM evidence on the location and number of Vo.r neuron terminations study (Shigenaga et al., 2000). on trigeminal motoneurons. We combined HRP injections into Yoshida et al. • Contacts between Sensory Neurons and Motoneurons J. Neurosci., August 15, 2001, 21(16):6298–6307 6305 Figure 8. Photomicrographs showing contacts made by a labeled Vo.r-dl Figure 9. Size distributions of counter-stained somata without contacts neuron CL1 on the soma (A) and the juxtasomatic region (B) of counter- (open columns) and with contacts ( filled columns) in the JC (A) and the JO stained motoneurons. Filled arrowheads denote contacts that are relatively (B) motor nucleus. The somata measured were randomly selected from in focus. Contacts marked with open arrowheads are out of focus but could sections in case Vo.r-dl neuron CL5 and in case Vo.r-vm neuron OP3. be identified by adjusting the focus. Scale bar, 20 m. single JC and JO -motoneurons with Nb injections into single Vo.r neuron and a motoneuron could be determined as 5.0 for JC Vo.r neurons. This particular combination allowed determination -motoneurons and as 4.0 for JO motoneurons. These values, of the number and location of contacts on the complete dendritic combined with a total number of JC or JO motoneurons obtained tree of either JC or JO -motoneurons. We observed that Vo.r-dl from each case, render such prediction that, on average, a Vo.r-dl neurons gave off 10 times more boutons than Vo.r-vm neurons, neuron terminates on 887 ( 151; range, 404–1225 neurons) JC indicating that the proportion of motoneurons receiving contacts motoneurons, which covers 54.2% ( 9.3%; range, 26.2–77.9%) is higher for JC than JO motoneurons. Interestingly, the average of the total number of JC motoneurons. On the other hand, on number of contacts made by Vo.r neurons on -motoneurons was average, a Vo.r-vm neuron ends on 111 ( 9; range, 85–139 almost the same between the two kinds of pairs. However, in a neurons) JO motoneurons, which covers 22.5% ( 1.5%; range, previous EM study (Shigenaga et al., 2000), we found that vesicle 17.9–26.7%) of the total number of JO motoneurons. This dif- number and density are significantly higher in synaptic boutons ference renders the prediction that the proportion of motoneu- from Vo.r-dl than Vo.r-vm neurons. These results suggest that the rons contacted is 2.4 times higher for JC than JO motoneurons. synaptic efficacy exerted by a Vo.r-dl neuron is stronger than that Together, the present results indicate that the net effects of by a Vo.r-vm neuron. Furthermore, on average, 17.2 and 16.1% of Vo.r neurons exerted on trigeminal motoneurons are much boutons from single Vo.r-dl and Vo.r-vm neurons contacted 13.2 greater for the JC than JO motoneuron pool and suggest that the and 5.3% of counter-stained JC and JO motoneurons (soma and strength of unitary inputs from single Vo.r neurons to single juxtasomatic regions) with 3.5 and 2.8 contacts per soma, respec- motoneurons is also stronger for JC motoneurons. tively. These results imply that the mean value of the number of Another important observation of the present study was that somata contacted is higher for JC than JO motoneurons. every labeled bouton from Vo.r neurons never made contacts with In addition, the present study made it possible to predict the different dendrites of the same motoneuron or with different number of motoneurons that receive contacts from the individual somata. This finding provides evidence that multiple contacts Vo.r neurons. An average number of contacts made between a made by a labeled bouton from Vo.r neurons observed in a 6306 J. Neurosci., August 15, 2001, 21(16):6298–6307 Yoshida et al. • Contacts between Sensory Neurons and Motoneurons previous EM study (Shigenaga et al., 2000) are made between the (Vmes), can excite JC motoneurons because their axon collaterals bouton and different motoneurons. terminate in the JC motor nucleus in addition to the supratri- geminal nucleus, intertrigeminal region, and Vp (Shigenaga et al., Comparisons of contacts made by Vo.r neurons and 1989), where excitatory premotoneurons are located (Turman muscle spindle afferents on JC motoneurons and Chandler, 1994; Kolta, 1997; Yoshida et al., 1998). Second, a Because recent studies established that jaw-muscle spindle affer- prior EM study (Bae et al., 1996) revealed that periodontal Vmes ents use glutamate as the transmitter (Chandler, 1989) and their afferent terminals make synaptic contacts with distal dendrites of terminals represent features common to excitatory synapses (Bae JC motoneurons. Third, Yoshida et al. (1998) demonstrated that et al., 1996; Luo and Dessem, 1999), comparisons of data on periodontal Vp neurons with their axons traveling in the trigemi- jaw-muscle spindle afferents with those of premotoneurons pre- no–thalamic tracts, which are presumed to receive input from sented here are important. In previous studies (Yabuta et al., Vmes periodontal afferents, issue axon collaterals in the JC motor 1996; Yoshida et al., 1999), we injected HRP into single jaw- nucleus. Finally, it has been reported that sensitivity of periodon- muscle spindle afferents and single JC -motoneurons and found tal mechanoreceptors depends on the amount to which the recep- that most contacts (90%) are within 600 m from the soma. The tors are stretched during tooth movement, i.e., thresholds fall number of contacts located within 200 m of the dendritic tree is progressively from the fulcrum to the apex (Cash and Linden, approximately two times higher for the Vo.r-dl neurons than 1982); and that periodontal afferent terminals from the Vmes are jaw-muscle spindle afferents (53.6 vs 24.2%). This reflects that concentrated to the base of the roots, whereas those from the peripherally induced IPSPs can be reversed by hyperpolarizing trigeminal ganglion (TG) are most numerous around the middle current or chloride ions injected into the soma (Shigenaga et al., of the roots (Byers and Dong, 1989), thus, indicating that thresh- 1988b). In addition, the number of contacts made on single JC olds of periodontal afferents are lower for Vmes than TG affer- -motoneurons is higher for Vo.r neurons than jaw-muscle spin- ents. In other words, activation of low-threshold periodontal dle afferents (5.0 vs 2.1) (Yabuta et al., 1996; Yoshida et al., 1999). afferents precedes that of higher-threshold ones when the teeth In contrast to Vo.r-dl neurons, no somatic contacts were found in come together or in contact with hard food. We concluded that 20 pairs of a jaw-muscle spindle afferent and a JC -motoneuron low-threshold Vmes periodontal afferents provide a positive force (Yabuta et al., 1996; Yoshida et al., 1999). However, 4% of feedback regulation of JC motoneurons, whereas activation of boutons from single jaw-muscle spindle afferents were found to high-threshold (not noxious) TG afferents arrests chewing via make contacts with counter-stained JC motoneuronal somata inhibitory premotoneurons like Vo.r neurons presented here. [data from a study of Yoshida et al. (1999)]. 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