Distribution of Gabaergic and Glycinergic Premotor Neurons Projecting to the Facial and Hypoglossal Nuclei in the Rat

______www.paper.edu.cn

THE JOURNAL OF COMPARATIVE NEUROLOGY 378:283–294 (1997)

Distribution of GABAergic and Glycinergic Premotor Neurons Projecting to the Facial and Hypoglossal Nuclei in the Rat

YUN-QING LI, MASAHIKO TAKADA, TAKESHI KANEKO, AND NOBORU MIZUNO* Department of Morphological Brain Science, Faculty of Medicine, Kyoto University, Kyoto 606-01, Japan

ABSTRACT The distribution of inhibitory premotor neurons for the facial and hypoglossal nuclei was examined in the lower brainstem of the rat. A retrograde axonal tracing method with the ﬂuorescent tracer, tetramethylrhodamine dextran amine (TMR-DA), was combined with immunoﬂuorescence histochemistry for glutamic acid decarboxylase (GAD), i.e., the enzyme involved in gamma-aminobutyric acid synthesis, or glycine. In the rats injected with TMR-DA unilaterally into the facial or hypoglossal nucleus, the distribution of TMR-DA-labeled neurons showing GAD-like immunoreactivity (GAD/TMR-DA neurons) was essentially the same as that of TMR-DA-labeled neurons displaying glycine-like immunoreactivity (Gly/ TMR-DA neurons). The distributions of GAD/TMR-DA and Gly/TMR-DA neurons in the rats injected with TMR-DA into the facial nucleus were also similar to those in the rats injected with TMR-DA into the hypoglossal nucleus. These neurons were seen most frequently in the lateral aspect of the pontine reticular formation, the supratrigeminal region, the dorsal aspect of the lateral reticular formation of the medulla oblongata, and the reticular regions around the raphe magnus nucleus and the gigantocellular reticular nucleus pars alpha, bilaterally with a slight dominance on the side ipsilateral to the injection site. A number of GAD/TMR-DA and Gly/TMR-DA neurons were also seen in the oral and interpolar subnuclei of the spinal trigeminal nucleus, bilaterally with a slight ipsilateral dominance. In the rats injected with TMR-DA into the facial nucleus, GAD/TMR-DA and Gly/TMR-DA neurons were also encountered in the paralemniscal zone of the midbrain tegmentum bilaterally with an apparent dominance on the side contralateral to the injection site. A large part of these inhibitory premotor neurons for the facial and hypoglossal nuclei and the excitatory ones may constitute premotor neuron pools common to the orofacial motor nuclei implicated in the control of integrated orofacial movements. J. Comp. Neurol. 378:283–294, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: face; tongue; reticular formation; brainstem; immunohistochemistry

The distribution of premotor neurons, which send their and glycinergic afferent ﬁbers are distributed within the axons to the facial and hypoglossal nuclei, have been facial and hypoglossal nuclei (Mugnaini and Oertel, 1985; examined by retrograde axonal tracing in the rat (Borke et Aldes et al., 1988; Fort et al., 1990). The distribution of al., 1983; Hinrichsen and Watson, 1983; Travers and inhibitory premotor neurons projecting to the facial and Norgren, 1983; Grzanna et al., 1987; Isokawa-Akesson and hypoglossal nuclei, however, have not been investigated Komisaruk, 1987; Aldes, 1990; Aldes et al., 1992; Manaker et al., 1992; Manaker and Tischler, 1993; Li et al., 1993a–c; Contract grant sponsor: Ministry of Education, Science, Sports, and Mogoseanu et al., 1994; Dobbins and Feldman, 1995; Culture of Japan; Contract grant numbers 0827906, 08458245. Ugolini, 1995; Yang et al., 1995) and in the cat (Tanaka et Dr. Yun-Qing Li is on leave from the Department of Anatomy, The Fourth al., 1978; Takeuchi et al., 1979; Itoh et al., 1983; Takada et Military Medical University, Xi’an, People’s Republic of China. al., 1984a,b; Fort et al., 1989; May et al., 1990; Ono et al., *Correspondence to: Dr. Noboru Mizuno, Department of Morphological Brain Science, Faculty of Medicine, Kyoto University, Kyoto 606-01, Japan. 1994). Both excitatory and inhibitory neurons must be E-mail: [email protected] contained in these premotor neurons projecting to the Received 3 June 1996; Revised 19 August 1996; Accepted 25 September facial and hypoglossal nuclei. In fact, many GABAergic 1996 r 1997 WILEY-LISS, INC. 中国科技论文在线______www.paper.edu.cn

284 Y.-Q. LI ET AL. systematically. In our previous study (Li et al., 1996), the then anesthetized deeply with an overdose of sodium distributions of gamma-aminobutyric acid (GABA)-ergic pentobarbital, and perfused transcardially with a volume and glycinergic premotor neurons projecting to the trigemi- of 100 ml of 0.9% saline, followed by a volume of 500 ml of nal motor nucleus were examined in the rat by a double- 0.1 M PB (pH 7.4) containing 4% paraformaldehyde, 0.25% labeling method that combined retrograde axonal tracing glutaraldehyde, and 0.2% picric acid. After the perfusion, with immunofluorescence histochemistry. Thus, the pre- the brains were removed immediately, placed in 0.1 M PB sent study was attempted primarily to examine the distri- (pH 7.4) containing 4% paraformaldehyde and 0.2% picric bution of GABAergic and glycinergic premotor neurons acid for 4–6 hours at 4°C and then saturated with 30% projecting to the facial and hypoglossal nuclei in the rat. A sucrose in 0.1 M PB (pH 7.4) at 4°C. Subsequently, the retrograde axonal tracing method with the fluorescent brainstems were cut serially into frontal sections 40-µm tracer, tetramethylrhodamine dextran amine (TMR-DA), thick on a freezing microtome. The sections were collected was combined with immunofluorescence histochemistry serially into three dishes containing 0.01 M phosphate for glutamic acid decarboxylase (GAD), i.e., the enzyme buffered saline (PBS; pH 7.3); each dish contained every involved in GABA synthesis, or glycine. Transported third section. TMR-DA was immunoreacted with a guinea pig anti- The sections in the first and the second dishes were TMR-DA IgG (Kaneko et al., 1996). GAD or glycine was rinsed in 0.01 M PBS (pH 7.3) and then processed for GAD immunoreacted with a sheep anti-GAD serum or a rabbit and glycine immunofluorescence histochemistry, respec- anti-glycine serum, respectively; the fluorescence dye used tively. The incubation medium used for dilution of the for visualization of GAD or glycine was fluorescein isothio- primary and the secondary antibodies was 0.01 M PBS (pH cyanate (FITC) or dichlorotriazinyl aminofluorescein 7.3) containing 2% normal donkey serum (NDS), 0.5% (DTAF), respectively. Triton X-100, 0.05% sodium azide (NaN3), and 0.25% l-car rageenan (NDS-PBS). For GAD immunofluorescence histochemistry, the sections were incubated overnight in NDS- MATERIALS AND METHODS PBS containing a sheep antiserum against GAD diluted at Adult male Wistar rats (Oriental Bioservice, Kyoto, 1:3,000 (a generous gift from Dr. I.J. Kopin; see Oertel et Japan) weighing 300–400 g were used in the present study. al., 1981) and 5 µg/ml guinea pig IgG against TMR-DA In 18 rats anesthetized by intraperitoneal injection of (Kaneko et al., 1996). The sections were then incubated for sodium pentobarbital (40 mg/kg body weight), single injec- 4 hours in NDS-PBS containing 10 µg/ml biotinylated tions of TMR-DA (MW 3,000, D-3308; Molecular Probes, donkey anti-sheep IgG (Chemicon) and for 2 hours with Eugen, OR) were made stereotaxically into the facial or 0.01 M PBS (pH 7.3) containing 0.3% Triton X-100, FITC- hypoglossal nucleus for retrograde axonal tracing (see labeled avidin D (Vector, Burlingame, CA) diluted at Kaneko et al., 1996). An approximate volume of 0.05–0.1 µl 1:2,000 and 10 µg/ml of TMR-labeled goat anti-guinea pig of a 10% solution of TMR-DA dissolved in 0.1 M phosphate IgG (Chemicon, Temecula, CA). For glycine immunofluores- buffer (PB; pH 7.4) was injected by pressure through a cence histochemistry, the sections were incubated over- glass micropipette (internal tip diameter 5 50–60 µm) night with NDS-PBS containing a rabbit antiserum against attached to a 1-µl Hamilton microsyringe. Each injection glycine (Chemicon) diluted at 1:2,000 and 5 µg/ml of the was made slowly over 20 minutes, and then the glass guinea pig antiserum against TMR (Kaneko et al., 1996). micropipette used for the TMR-DA injection was kept in The sections were then incubated for 4 hours with DTAF- place for additional 20 minutes. After 3 days, the rats were labeled donkey anti-rabbit IgG (Chemicon) and TMR- again anesthetized and then injected stereotaxically with labeled goat anti-guinea pig IgG (Chemicon), both of which a volume of 10 µl of 1% colchicine (Sigma, St. Louis, MO) were diluted to 10 µg/ml in NDS-PBS. dissolved in 0.9% saline into the lateral ventricle. Subse- After immunofluorescence staining, the sections were quently, the rats were allowed to survive for 36–48 hours, mounted onto clean glass slides, air dried at room tempera-

Abbreviations

Aq cerebral aqueduct NTS nucleus of the solitary tract C1 1st cervical segment of the spinal cord PL paralemniscal zone C5 5th cervical segment of the spinal cord PN pontine nuclei CC central canal PTA pretectal area CG central gray py pyramidal tract cp cerebral peduncle R red nucleus Dk nucleus of Darkschewitsch SC superior colliculus DR dorsal raphe nucleus scp superior cerebellar peduncle EW nucleus of Edinger–Westphal SN substantia nigra f exiting root of the facial nerve SO superior olivary complex g genu of the root of the facial nerve SupV supratrigeminal region GiA gigantocellular reticular nucleus pars alpha t spinal tract of the trigeminal nerve h root of the hypoglossal nerve III oculomotor nucleus IC inferior colliculus IV trochlear nucleus InC interstitial nucleus of Cajal Vc caudal subnucleus of the spinal trigeminal nucleus IO inferior olivary complex Vi interpolar subnucleus of the spinal trigeminal nucleus IP interpeduncular nucleus Vm trigeminal motor nucleus KF nucleus of Ko¨lliker–Fuse Vo oral subnucleus of the spinal trigeminal nucleus LPB lateral parabrachial nucleus Vp principal sensory trigeminal nucleus LRF lateral reticular formation VII facial nucleus MPB medial parabrachial nucleus X dorsal motor nucleus of the vagus nerve MRF medial reticular formation XII hypoglossal nucleus NRM nucleus raphe magnus 中国科技论文在线______www.paper.edu.cn

INHIBITORY NEURONS TO VII AND XII CRANIAL NUCLEI 285 ture, and coverslipped with PBS containing 50% glycerol each) in 0.01 M PBS (pH 7.3) between each step of the and 2.5% triethylenediamine. Neuronal cell bodies labeled immunostaining. retrogradely with TMR-DA, GAD-like immunoreactive The sections in the third dish were mounted onto cell bodies labeled with FITC, and glycine-like immunore- gelatin-coated glass slides and then stained with 1% cresyl active ones labeled with DTAF were observed with an violet. These sections were used to examine the anatomical epifluorescence microscope (Axiophoto; Zeiss, Oberkochen, sites of distribution of GAD-like and glycine-like immuno- Germany) under appropriate filters for TMR-DA (excita- reactive cells in the adjacent sections stained immunohis- tion 5 540–552 nm; emission $ 590 nm) and for FITC and tochemically. DTAF (excitation 5 450–490 nm; emission 5 515–565 nm). In control immunostaining for GAD, the anti-GAD se- The sections in the third dish were mounted onto rum was replaced with a preimmune sheep serum diluted gelatin-coated glass slides and stained with 1% cresyl at 1:1,000. In control immunostaining for glycine, the violet. A number of cresyl violet-stained sections through antiglycine serum was preabsorbed with an excess amount lower brainstem levels were selected, and then large of glycine or glycine–bovine serum albumin (BSA) conju- projection drawings (340) were prepared. In the sections gate. The glycine–BSA conjugate was made as follows: 2 stained by immunofluorescence histochemistry, the sites of mg (30.3 nmol) of BSA and 9.1 µl (910 nmol) of 0.1 M tracer injection and the locations of neuronal cell bodies glycine were mixed in 1 ml of 0.1 M PB (pH 7.4) and labeled with TMR-DA were identified under the epifluores- conjugated by incubation for 2 hours at room temperature cence microscope (4 3 10) and charted on the projection after an addition of 25 µl of 5% glutaraldehyde. The drawings of the adjacent cresyl violet-stained sections as conjugate was dialyzed against PBS before use. The anti- exactly as possible by referring to large blood vessels and glycine serum diluted at 1:1,000 in NDS-PBS was incu- other prominent landmarks in the sections. Neuronal cell bated with 0.1–0.5 M glycine or 0.2–1 mg BSA-equivalent/ bodies double-labeled with TMR-DA/FITC or TMR-DA/ ml glycine-BSA conjugate for 2 hours at room temperature DTAF were then identified among the cell bodies single- before immunostaining. labeled with TMR-DA at a higher magnification (20 3 10) to record them in the projection drawings. Subsequently, RESULTS the data were reconstructed onto tracings of sets of serial sections (Figs. 3, 4, 6, 8); in each rat, the locations of Distribution of GAD-like and glycine-like neuronal cell bodies double-labeled with TMR-DA/FITC immunoreactive neurons and TMR-DA/DTAF were reconstructed separately from in the lower brainstem single-labeled ones in a single set of projection drawings of Immunohistochemical staining for GAD and glycine was a series of serial sections (Figs. 6, 8). The nomenclature done by the ABC method (Hsu et al., 1981). A considerable and boundaries of the structures in the lower brainstem number of neurons with GAD-like immunoreactivity and were basically taken from the atlas of Paxinos and Watson those with glycine-like immunoreactivity were widely (1986); the brainstem reticular formation was divided into scattered in the lower brainstem (Fig. 1a,c), as was ob- the lateral and medial parts according to Holstege and served in our previous study (Li et al., 1996). Briefly, they Kuypers (1977). were seen most frequently in the reticular formation of the To observe the morphological features of GAD-like and pons and medulla oblongata, the supratrigeminal region, glycine-like immunoreactive neurons under brightfield the paralemniscal zone, the superior olivary complex, and illumination, immunohistochemical staining for GAD and the prepositus hypoglossi nucleus. A number of GAD-like glycine was also performed according to the ABC method immunoreactive neurons were also distributed in the (Hsu et al., 1981). Two rats underwent the colchicine nuclei of the cranial nerves, such as the oculomotor treatment and transcardial perfusion. The brainstems nucleus, the trigeminal motor nucleus, the sensory trigemi- were cut serially into frontal sections 40-µm thick on the nal nuclei, the cochlear nuclei, the vestibular nuclei, and freezing microtome and collected into three dishes contain- the nucleus of the solitary tract. The dorsal column nuclei ing 0.01 M PBS (pH 7.3). The sections in the first and the also contained GAD-like immunoreactive neurons. Glycine- second dishes were processed for GAD and glycine immuno- like immunoreactive neurons were particularly marked in histochemistry, respectively. Briefly, for GAD immunohis- the superior olivary complex, the nucleus of the trapezoid tochemistry, the sections were incubated overnight with body, the cochlear nuclei, the vestibular nuclei, and the NDS-PBS containing the sheep antiserum against GAD prepositus hypoglossi nucleus, although they were distrib- (see Oertel et al., 1981) diluted at 1:3,000 and then with uted in virtually all nuclei of the lower brainstem. The biotinylated donkey anti-sheep IgG (Chemicon) diluted at GAD-like or glycine-like immunoreactive neurons were oval, 10 µg/ml in NDS-PBS for 4 hours. For glycine immunohis- triangular, or fusiform in shape; in most of them, the longest tochemistry, the sections were incubated overnight with soma diameter was 8–25 µm for GAD and 8–30 µm for glycine. NDS-PBS containing the rabbit antiserum against glycine However, in some regions, particularly in the regions around (Chemicon) diluted at 1:2,000 and then with 10 µg/ml of the gigantocellular reticular nucleus pars alpha, a number of biotinylated goat anti-rabbit IgG (Vector) for 4 hours. fusiform neurons with GAD-like immunoreactivity had These sections processed for GAD or glycine immunohisto- larger soma diameters reaching up to 35 µm. chemistry were then incubated with ABC (Vector) diluted The existence of many fine axonal components showing at 1:200 in 0.01 M PBS (pH 7.3) containing 0.3% Triton GAD-like or glycine-like immunoreactivity was also con- X-100 for 2 hours. Subsequently, the sections were placed firmed in the facial and hypoglossal nuclei (data not for 20–30 minutes in 0.05 M Tris-HCl buffer (pH 7.6) shown). containing 0.04% diaminobenzidine tetrahydrochloride In control immunostaining, when the anti-GAD serum (Dojin, Kumamoto, Japan) and 0.001% H2O2. All immuno- was replaced with preimmune sheep serum diluted at histochemical procedures were carried out at room tem- 1:1,000, no immunoreactivity was found in the sections perature; the sections were rinsed three times (10 minutes (Fig. 1b). When the antiglycine serum diluted at 中国科技论文在线______www.paper.edu.cn

286 Y.-Q. LI ET AL.

Fig. 2. The sites of tetramethylrhodamine dextran amine (TMR- DA) injection in the facial nucleus of a rat (R6; a) and in the hypoglossal nucleus of another rat (R10; b). Scale bars 5 250 µm. Fig. 1. Glutamic acid decarboxylase (GAD)-like (a) and glycine- like (c) immunoreactivities in the lateral part of the medullary reticular formation. When the anti-GAD serum was replaced with However, the lower brainstem areas containing the TMR- preimmune sheep serum, no immunoreactivity was seen in the lateral DA-labeled neuronal cell bodies were virtually identical part of the medullary reticular formation (b). When the antiglycine serum was preabsorbed with an excess amount of glycine, no glycine- among the rats of each group. Therefore, to avoid unneces- like immunoreactivity was seen in the lateral part of the medullary sary repetition, only the typical ﬁndings observed in a reticular formation (d). Scale bar 5 50 µm. representative rat of each group will be described; in these representative rats (R6 and R10), the number of TMR-DA- labeled neurons was largest in each group. After TMR-DA injection into the facial nucleus (Fig. 2a), 1:1,000 in NDS-PBS was preincubated with 0.5 M glycine many TMR-DA-labeled neuronal cell bodies were seen in or 0.2 mg/ml glycine–BSA conjugate, no glycine-like immu- the tegmental regions of the lower brainstem (Fig. 3). At noreactivity was found in the sections (Fig. 1d). midbrain levels, many labeled neurons were seen in the Distribution of neurons labeled retrogradely medial aspect of the pretectal area and the paralemniscal with TMR-DA injected into the facial zone close to the dorsomedial border of the medial lemniscus, bilaterally with an apparent predominance on the side or hypoglossal nucleus contralateral to the injection site. Labeled neuronal cell In the present study, the intense deposition area of bodies were also distributed in the perioculomotor regions, TMR-DA at the center of the injection site and the diffusion including the nucleus of Edinger–Westphal, the nucleus of area surrounding the center of the injection site were Darkschewitsch, the interstitial nucleus of Cajal, the considered as the maximum probable sites of termination ventral part of the periaqueductal gray, the dorsal raphe for neurons labeled retrogradely with the injected TMR- nucleus, and the oculomotor nucleus, and in the mesence- DA. The sites of TMR-DA injection were successfully phalic reticular formation, bilaterally with an ipsilateral centered in the facial or hypoglossal nucleus in 12 rats dominance. Labeled neurons within the oculomotor nucleus (Fig. 2). The injection sites in six rats of each group were were smaller than the oculomotor motoneurons. A small not identical, and the number of TMR-DA-labeled neurons number of neurons in the red nucleus and the deep part of differed according to the extent of the injection site. the superior colliculus also were labeled contralaterally to 中国科技论文在线______www.paper.edu.cn

INHIBITORY NEURONS TO VII AND XII CRANIAL NUCLEI 287

Fig. 3. Distribution of neuronal cell bodies that were labeled and medulla oblongata. Each number indicates the position of the retrogradely with TMR-DA injected into the facial nucleus of a rat section in a series of serial 40-µm-thick sections that are arranged (R6). The injection site is blackened. Neuronal cell bodies labeled with rostrocaudally; the number of a section through a level of the obex is TMR-DA (ﬁlled circles) are plotted in a one-to-one fashion on a series of indicated by a 0. projection drawings of frontal sections through the midbrain, pons, the injection site; few, if any, labeled neurons were found in the gigantocellular reticular nucleus pars alpha, bilater- these regions ipsilateral to the injection site. At pontine ally with a slight ipsilateral dominance. Only a few labeled levels, many labeled neurons were seen in the parabra- neurons were distributed in the medial part of the reticu- chial region, including the medial and lateral parabrachial lar formation. A number of labeled neurons were also seen nuclei and the nucleus of Ko¨lliker–Fuse, the supratrigemi- in the sensory trigeminal nuclei, especially in the oral and nal region, the lateral part of the pontine reticular forma- interpolar subnuclei of the spinal trigeminal nucleus ipsi- tion, and the ventral part of the reticular formation around lateral to the injection site. A small number of labeled the raphe magnus nucleus, bilaterally with a slight domi- neuronal cell bodies were also distributed in the intermedi- nance on the side ipsilateral to the injection site. At levels ate zone of the upper and middle cervical segments of the of the medulla oblongata, labeled neuronal cell bodies were spinal cord. encountered most frequently in the lateral and dorsal After TMR-DA injection into the hypoglossal nucleus parts of the reticular formation and the ventral parts of the (Fig. 2b), many labeled neuronal cell bodies were seen in reticular formation around the raphe magnus nucleus and the pontine and medullary reticular formation, bilaterally 中国科技论文在线______www.paper.edu.cn

288 Y.-Q. LI ET AL.

Fig. 4. Distribution of neuronal cell bodies that were labeled retrogradely with TMR-DA injected into the hypoglossal nucleus of a rat (R10). The injection site is blackened. Neuronal cell bodies labeled with TMR-DA (ﬁlled circles) are plotted in a one-to-one fashion, as in Figure 3. with a slight dominance on the side ipsilateral to the zone of the upper and middle cervical segments of the injection site (Fig. 4). The pattern of distribution of these spinal cord. labeled neuronal cell bodies in the pontine and medullary reticular formation was essentially the same as that GAD-like and glycine-like immunoreactive observed after TMR-DA injection into the facial nucleus. neurons labeled retrogradely with TMR-DA At mesencephalic levels, however, only a small number of injected into the facial or labeled neurons were scattered in the mesencephalic reticular formation, the periaqueductal gray, and the dorsal hypoglossal nucleus raphe nucleus. No labeled neurons were found in the In the rats injected with TMR-DA into the facial nucleus pretectal area or the red nucleus. Few, if any, labeled (Fig. 2a), some of TMR-DA-labeled neurons had GAD-like neurons were observed in the deep part of the superior or glycine-like immunoreactivity (Fig. 5). Both TMR-DA- colliculus or the paralemniscal zone. At pontine levels, labeled neurons showing GAD-like immunoreactivity many labeled neurons were seen in the parabrachial (GAD/TMR-DA neurons) and TMR-DA-labeled neurons region, especially in the nucleus of Ko¨lliker–Fuse ipsilat- displaying glycine-like immunoreactivity (Gly/TMR-DA eral to the injection site. A number of labeled neurons were neurons) were found mainly in the paralemniscal zone, the seen in the sensory trigeminal nuclei and the intermediate lateral part of the pontine reticular formation, the supra- 中国科技论文在线______www.paper.edu.cn

INHIBITORY NEURONS TO VII AND XII CRANIAL NUCLEI 289

Fig. 5. Sections containing neurons labeled with TMR-DA injected immunoreactive neuron labeled with TMR-DA in the raphe magnus into the facial nucleus (a,b) are immunostained for GAD (a8) or glycine nucleus. Arrows in a and b indicate neurons single-labeled with (b8). Double arrowheads in a and a8 point to GAD-like immunoreactive TMR-DA. Arrowheads in a8 and b8 indicate TMR-DA-negative neurons neuron labeled with TMR-DA in the gigantocellular reticular nucleus showing GAD-like (a8) or glycine-like (b8) immunoreactivity. Scale pars alpha. Double arrowheads in b and b8 indicate glycine-like bar 5 25 µm.

trigeminal region, the dorsal part of the lateral reticular interpolar subnuclei of the spinal trigeminal nucleus, formation of the medulla oblongata, the ventral parts of bilaterally with a slight dominance on the side ipsilateral the medullary reticular formation around the raphe mag- to the injection site (Fig. 8). nus nucleus and the gigantocellular reticular nucleus pars alpha, and the oral and interpolar subnuclei of the spinal trigeminal nucleus (Fig. 6). In the paralemniscal zone, DISCUSSION GAD/TMR-DA neurons were seen bilaterally with an In the present study, the distribution of premotor neu- apparent dominance on the side contralateral to the rons for the facial and hypoglossal nuclei in the lower injection site. In the other regions, GAD/TMR-DA neurons brainstem was examined in the rat by the retrograde were seen bilaterally with a slight dominance on the side labeling method with TMR-DA. Fluorochrome-conjugated ipsilateral to the injection site. dextran amines, including TMR-DA, were initially intro- Some of TMR-DA-labeled neurons observed in the rats duced as ﬂuorescent anterograde tracers in the central injected with TMR-DA into the hypoglossal nucleus (Fig. nervous system. However, TMR-DA can also be used as a 2b) also showed GAD-like or glycine-like immunoreactiv- very sensitive retrograde tracer (Kaneko et al., 1996). ity (Fig. 7), although the numbers of these GAD/TMR-DA The facial and hypoglossal nuclei of the rat are composed and Gly/TMR-DA neurons were smaller than those found of a number of subnuclei (Krammer et al., 1979; Watson et in the rats injected with TMR-DA into the facial nucleus al., 1982; Hinrichsen and Watson, 1984; Friauf and Her- (compare Fig. 8 with Fig. 6). Both of these GAD/TMR-DA bert, 1985; Klein and Rhoades, 1985; Semba and Egger, and Gly/TMR-DA neurons were found mainly in the dorsal 1986; Uemura-Sumi et al., 1988; Tsai et al., 1993; Aldes, part of the lateral reticular formation of the medulla 1995; Dobbins and Feldman, 1995), and the locations of oblongata and in the supratrigeminal region, the lateral premotor neurons projecting to different subdivisions are part of the pontine reticular formation, the reticular more or less different from each other (Hinrichsen and regions around the raphe magnus nucleus and the giganto- Watson, 1983; Travers and Norgren, 1983; Isokawa- cellular reticular nucleus pars alpha, and the oral and Akesson and Komisaruk, 1987; Dobbins and Feldman, 中国科技论文在线______www.paper.edu.cn

290 Y.-Q. LI ET AL.

Fig. 6. Distribution of TMR-DA-labeled neuronal cell bodies show- into the facial nucleus (see Figs. 2a, 3). The injection site is blackened. ing GAD-like immunoreactivity (ﬁlled triangles) or glycine-like immu- The double-labeled neuronal cell bodies are plotted in a one-to-one noreactivity (open triangles) in R6, which was injected with TMR-DA fashion, as in Figure 3.

1995). In the present study, however, the TMR-DA injec- The present study has also shown that some of the tions were attempted to cover the entire extent of the facial neuronal cell bodies labeled retrogradely with TMR-DA or hypoglossal nucleus. The present ﬁndings concerning injected into the facial or hypoglossal nucleus are GAD- the distribution of premotor neurons for the facial and like or glycine-like immunoreactive. The areas of the lower hypoglossal nuclei were compatible with almost all data brainstem containing GAD-like immunoreactive TMR-DA- reported previously in the rat (Borke et al., 1983; Hinrich- labeled neurons (GAD/TMR-DA neurons) greatly over- sen and Watson, 1983; Isokawa-Akesson and Komisaruk, lapped the areas containing glycine-like immunoreactive 1987; Aldes, 1990; Ter Horst et al., 1991; Aldes et al., 1992; TMR-DA-labeled neurons (Gly/TMR-DA neurons). Thus, Manaker et al., 1992; Manaker and Tischler, 1993; Li et both GAD-like and glycine-like immunoreactivities may al., 1993a–c; Mogoseanu et al., 1994; Dobbins and Feld- coexist in some inhibitory premotor neurons projecting to man, 1995; Ugolini, 1995; Yang et al., 1995) and cat the facial or hypoglossal nucleus (see Todd and Sullivan, (Tanaka et al., 1978; Takeuchi et al., 1979; Itoh et al., 1983; 1990; Maxwell et al., 1995; Davanger, 1996; Lahjouji et al., Takada et al., 1984a,b; May et al., 1990; Ono et al., 1994). 1996; Moore et al., 1996; Popratiloff et al., 1996; Todd et al., 中国科技论文在线______www.paper.edu.cn

INHIBITORY NEURONS TO VII AND XII CRANIAL NUCLEI 291

Fig. 7. Sections containing neurons labeled with TMR-DA injected the dorsolateral part of the medullary reticular formation. Arrows in a into the hypoglossal nucleus (a,b) are immunostained for GAD (a8)or and b indicate neurons single-labeled with TMR-DA. Arrowheads in a8 glycine (b8). Double arrowheads in a and a8 point to GAD-like and b8 indicate TMR-DA-negative neurons showing GAD-like (a8)or immunoreactive neuron labeled with TMR-DA in the dorsolateral part glycine-like (b8) immunoreactivity. Asterisks in a and a8 indicate the of the medullary reticular formation. Double arrowheads in b and b8 same blood vessel. Scale bar 5 25 µm. indicate glycine-like immunoreactive neuron labeled with TMR-DA in

1996). At least some of the GAD/TMR-DA and Gly/ Edwards, 1978; Kume et al., 1978). Such direct projections TMR-DA neurons observed in the present study were from the paralemniscal zone to the facial nucleus have also GABAergic and glycinergic inhibitory premotor neurons been observed in the rat (Hinrichsen and Watson, 1983; participating in synaptic actions on facial and hypoglossal Isokawa-Akesson and Komisgruck, 1987) and in the opos- motoneurons, although local interneurons within and sum (Panneton and Martin, 1978, 1979, 1983). More around the facial and hypoglossal nuclei might also be the recently, both monosynaptic excitatory and inhibitory post- postsynaptic targets for some of the GAD/TMR-DA and synaptic potentials were recorded from motoneurons inner- Gly/TMR-DA neurons. vating the extrinsic and intrinsic pinna muscles by electri- In the rats injected with TMR-DA into the facial nucleus, cal stimulation of the contralateral paralemniscal zone in a significant number of GAD/TMR-DA and Gly/TMR-DA the cat (May et al., 1990). In the rat, 10.9% of GABA-like neurons were seen in the paralemniscal zone contralateral immunoreactive neurons in the paralemniscal zone were to the injection site, suggesting that both GABAergic and reported to be retrogradely labeled with horseradish peroxi- glycinergic neurons in the paralemniscal zone project dase injected contralaterally into the medial part of the directly to the facial nucleus. The paralemniscal zone, a facial nucleus (Chen et al., 1995). The present study has mesencephalic tegmental zone along the dorsomedial bor- confirmed and extended these previous observations, indi- der of the medial lemniscus, was identified in the cat as a cating that not only GABAergic but also glycinergic neu- major site of origin of mesencephalic projection fibers to rons in the paralemnisal zone send inhibitory projection the facial nucleus (Henkel and Edwards, 1978). This zone fibers to the facial nucleus. receives afferent fibers from the superior colliculus (Hen- In the rats injected with TMR-DA into the facial nucleus, kel and Edwards, 1978; Stein et al., 1984; see also Gray- the pretectal area contralateral to the injection site also biel, 1977) and periaqueductal gray (Henkel, 1981) and contained a cluster of TMR-DA-labeled neurons in its sends projection fibers to the medial part of the facial medial part. However, no TMR-DA-labeled neurons in the nucleus bilaterally with a clear contralateral dominance pretectal area showed either GAD-like or glycine-like immuno- (Henkel and Edwards, 1978; Takeuchi et al., 1979). The reactivity. It has been assumed that the pretectofacial medial part of the facial nucleus where the projection fibers arising from the pretectal olivary nucleus in the cat fibers from the paralemniscal zone terminate, contains innervate the orbicularis oculi muscle and constitute a link motoneurons innervating the pinna muscles (Henkel and in the arc of the visually triggered blink reflex (e.g., dazzle 中国科技论文在线______www.paper.edu.cn

292 Y.-Q. LI ET AL.

Fig. 8. Distribution of TMR-DA-labeled neuronal cell bodies show- into the hypoglossal nucleus (see Figs. 2b, 4). The injection site is ing GAD-like immunoreactivity (filled triangles) or glycine-like immu- blackened. The double-labeled neuronal cell bodies are plotted in a noreactivity (open triangles) in R10, which was injected with TMR-DA one-to-one fashion, as in Figure 3. or menace reflex; Itoh et al., 1983). Thus, it is interesting to TMR-DA injected into the facial nucleus most frequently note that the paralemniscal zone sending projection fibers showed GAD-like or glycine-like immunoreactivity in the to the facial motoneurons that innervate the pinna muscles lateral part of the reticular formation in the pons and contains inhibitory premotor neurons for the facial nucleus, medulla oblongata. whereas the pretectal area sending projection fibers to the Hypoglossal motoneurons receive indirect inputs from facial motoneurons innervating the orbicularis oculi muscle the trigeminal, glossopharyngeal, and superior laryngeal contains no inhibitory premotor neurons for the facial nerves and from the brain areas, including the cerebral nucleus. cortex and the brainstem regions with pattern/rhythm The lateral part of the pontomedullary reticular forma- generators for respiration, mastication, and deglutition tion, especially the parvicellular reticular formation, con- (for review, see Lowe, 1981; Travers and Jackson, 1992; tains many premotor neurons for the facial nucleus Ono et al., 1994); the premotor neurons mediating these (Holstege and Kuypers, 1977; Holstege et al., 1977; inputs to hypoglossal motoneurons are located mainly in Takeuchi et al., 1979; Panneton and Martin, 1983; Takada the lateral part of the medullary reticular formation et al., 1984a; Travers and Norgren, 1983; Isokawa- (Hostege and Kuypers, 1977; Holstege et al., 1977; Borke Akesson and Komisarum, 1987; Ter Horst et al., 1991; Li et et al., 1983; Travers and Norgren 1983; Takada et al., al., 1993a–c; Mogoseanu et al., 1994). In the present study, 1984b; Borke and Nau, 1985; Ter Holstege et al., 1991; Ono neuronal cell bodies that were labeled retrogradely with et al., 1994). Direct excitatory projections from the giganto- 中国科技论文在线______www.paper.edu.cn

INHIBITORY NEURONS TO VII AND XII CRANIAL NUCLEI 293 cellular reticular nucleus to the hypoglossal nucleus (Yang reticulo-hypoglossal axon terminals anterogradely labeled with horse- et al., 1995) and direct serotoninergic projections from the radish peroxidase. Brain Res. 337:127–132. raphe nuclei to the hypoglossal nucleus (Li et al., 1993c; Borke, R.C., M.E. Nau, and R.L. Ringler, Jr. (1983) Brain stem afferents of hypoglossal neurons in the rat. Brain Res. 269:47–55. Manaker and Tischler, 1993) have also been reported in Chen, X.H., M. Itoh, T. Miki, M. Ichikawa, Y. Fujimoto, W. Sun, and Y. the rat. In the present study, a number of GAD/TMR-DA Takeuchi (1995) Midbrain paralemniscal projections to the facial and Gly/TMR-DA neurons were observed in these regions nucleus: An anatomical and immunohistochemical study. Okajimas of the rats injected with TMR-DA into the hypoglossal Folia Anat. Jpn. 72:69–80. nucleus. Davanger, S. (1996) Colocalization of amino acid signal molecules in In summary, in rats injected with TMR-DA into the neurons and endocrine cells. Anat. Embryol. 194:1–12. facial nucleus and those injected with TMR-DA into the Dobbins, E.G., and J.L. Feldman (1995) Differential innervation of pro- truder and retractor muscles of the tongue in rat. J. Comp. Neurol. hypoglossal nucleus, GAD/TMR-DA and Gly/TMR-DA neu- 357:376–394. rons were observed mainly in the lateral parts of the Fort, P., K. Sakai, P.-E´ . Luppi, D. Salvert, and M. Jouvet (1989) Monoamin- pontomedullary reticular formation, especially in the supra- ergic, peptidergic, and cholinergic afferents to the cat facial nucleus as trigeminal region and the parvicellular reticular forma- evidenced by a double immunostaining method with unconjugated tion and in the medullary reticular formation around the cholera toxin as a retrograde tracer. J. Comp. Neurol. 283:285–302. raphe magnus nucleus and the gigantocellular reticular Fort, P., P.-H. Luppi, R. Wenthold, and M. Jouvet (1990) Neurones nucleus pars alpha; these neurons were distributed bilater- immunoreáctifs a` la glycine dans le bulbe rachidien du chat. C. R. Acad. Sci. Paris 311 Se´rie III:205–212. ally with a slight dominance on the side ipsilateral to the Friauf, E., and H. Herbert (1985) Topographic organization of facial TMR-DA injection. The supratrigeminal region is a premo- motoneurons to individual pinna muscles in rat (Rattus rattus) and bat tor neuron pool for the trigeminal motor nucleus; in (Rousettus aegyptiacus). J. Comp. Neurol. 240:161–170. particular, it contains inhibitory neurons projecting to Goldberg, L.J., and Y. Nakamura (1968) Lingually induced inhibition of jaw-closing motoneurons (Goldberg and Nakamura, 1968; masseteric motoneurones. Experientia 24:371–373. Kidokoro et al., 1968; Mizuno et al., 1978, 1983; Ohta and Graybiel, A.M. (1977) Direct and indirect preoculomotor pathways of the Moriyama, 1986; Li et al., 1996). The patterns of distribu- brainstem: An autoradiographic study of the pontine reticular formation of GAD/TMR-DA and Gly/TMR-DA neurons in the tion in the cat. J. Comp. Neurol. 175:37–78. Grzanna, R., W.K. Chee, and E.W. Akeyson (1987) Noradrenergic projec- parvicellular reticular formation and the regions around tions to brainstem nuclei: Evidence for differential projections from the raphe magnus nucleus and the gigantocellular reticu- noradrenergic subgroups. J. Comp. Neurol. 263:76–91. lar nucleus pars alpha were also similar to those of Henkel, C.K. (1981) Afferent sources of a lateral midbrain tegmental zone GAD-like and glycine-like immunoreactive neurons project- associated with the pinnae in the cat as mapped by retrograde transport ing to the trigeminal motor nucleus (compare the present of horseradish peroxidase. J. Comp. Neurol. 203:213–226. data with those of the previous studies; for rat: Li et al., Henkel, C.K., and S.B. Edwards (1978) The superior colliculus control of 1996; for guinea pig: Turman and Chandler, 1994). Thus, it pinna movements in the cat: Possible anatomical connections. J. Comp. Neurol. 182:763–776. was assumed that GABAergic and glycinergic inhibitory Hinrichsen, C.F.L., and C.D. Watson (1983) Brain stem projections to the neurons with excitatory neurons in these regions might facial nucleus of the rat. Brain Behav. Evol. 22:153–163. constitute premotor neuron pools common to the orofacial Hinrichsen, C.F.L., and C.D. Watson (1984) The facial nucleus of the rat: motor nuclei. Representation of facial muscles revealed by retrograde transport of horseradish peroxidase. Anat. Rec. 209:407–415. Holstege, G., and H.G.J.M. Kuypers (1977) Propriobulbar fibre connections ACKNOWLEDGMENTS to the trigeminal, facial and hypoglossal motor nuclei. I. An anterograde degeneration study in the cat. Brain 100:239–264. We are grateful for the photographic help of Mr. Akira Holstege, G., H.G.J.M. Kuypers, and J.J. Dekker (1977) The organization of Uesugi. We also express our gratitude for the support of the bulbar fibre connections to the trigeminal, facial and hypoglossal Drs. Satoru Fukuchi, Ritsu Hayashi, Sohzaburo Hayashi, motor nuclei. II. An autoradiographic tracing study in cat. Brain Mizuho Katsurada, Yutaka Kitani, Toshihiko Kuroda, 100:265–286. Keiko Kumagai, Hiroshi Matsubara, Hiroshi Matsushima, Hsu, S.-M., L. Raine, and H. Fanger (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison be- Chisato Minakuchi, Gonpei Niwa, Hajime Oda, Masahiko tween ABC and unlabeled antibody (PAP) procedures. J. Histochem. Ohbayashi, Sei-ichi Ohbayashi, Hiroyasu Ohtsuka, Shigeo Cytochem. 29:577–580. Tamaki, Eizo Watanabe, Kazuo Yoshino, and Toshiaki Isokawa-Akesson, M., and B.R. Komisaruk (1987) Difference in projections Yoshino. This work was supported in part by Grants-in- to the lateral and medial facial nucleus: Anatomically separate pathways Aid (0827906 and 08458245) from the Ministry of Educa- for rhythmical vibrissa movement in rats. Exp. Brain Res. 65:385–398. tion, Science, Sports and Culture of Japan. Itoh, K., M. Takada, Y. Yasui, and N. Mizuno (1983) A pretectofacial projection in the cat: Possible link in the visually triggered blink reflex pathways. Brain Res. 274:332–335. LITERATURE CITED Kaneko, T., K. Saeki, T. Lee, and N. Mizuno (1996) Improved retrograde axonal transport and subsequent visualization of tetramethylrhoda- Aldes, L.D. (1990) Topographically organized projections from the nucleus mine (TMR)-dextran amine by means of an acidic injection vehicle and subceruleus to the hypoglossal nucleus in the rat: A light and electron antibodies against TMR. J. Neurosci. Methods 65:157–165. microscopic study with complementary axonal transport techniques. J. Kidokoro, Y., K. Kubota, S. Shuto, and R. Sumino (1968) Possible interneu- Comp. Neurol. 302:643–656. rons responsible for reflex inhibition of motoneurons of jaw-closing Aldes, L.D. (1995) Subcompartmental organization of the ventral (protru- muscles from the inferior dental nerve. J. Neurophysiol. 31:709–716. sor) compartment in the hypoglossal nucleus of the rat. J. Comp. Klein, B.G., and R.W. Rhoades (1985) Representation of whisker follicle Neurol. 353:89–108. intrinsic musculature in the facial motor nucleus of the rat. J. Comp. Aldes, L.D., R.B. Chronister, and L.A. Marco (1988) Distribution of Neurol. 232:55–69. glutamic acid decarboxylase and gamma-aminobutyric acid in the Krammer, E.B., T. Rath, and M.F. Lischka (1979) Somatotopic organization hypoglossal nucleus in the rat. J. Neurosci. Res. 19:343–348. of the hypoglossal nucleus: A HRP study in the rat. Brain Res. Aldes, L.D., M.E. Chapman, R.B. Chronister, and J.W. Haycock (1992) 170:533–537. Sources of noradrenergic afferents to the hypoglossal nucleus in the rat. Kume, M., M. Uemura, K. Matsuda, R. Matsushima, and N. Mizuno (1978) Brain Res. Bull. 29:931–942. Topographical representation of peripheral branches of the facial nerve Borke, R.C., and M.E. Nau (1985) The ultrastructural identification of within the facial nucleus: A HRP study in the cat. Neurosci. Lett. 8:5–8. 中国科技论文在线______www.paper.edu.cn

294 Y.-Q. LI ET AL.

Lahjouji, F., A. Barbe, G. Chazal, and H. Bras (1996) Evidence for trigeminal, facial and hypoglossal nuclei in the opossum. A study using colocalization of GABA and glycine in afferents to retrogradely labelled axonal transport techniques. Brain Res. 168:493–511. rat abducens motoneurones. Neurosci. Lett. 206:161–164. Panneton, W.M., and G.F. Martin (1983) Brainstem projections to the facial Li, Y.-Q., M. Takada, and N. Mizuno (1993a) Premotor neurons projecting nucleus of the opossum. A study using axonal transport techniques. simultaneously to two orofacial motor nuclei by sending their branched Brain Res. 267:19–33. axons. A study with a fluorescent retrograde double-labeling technique Paxinos, G., and C. Watson (1986) The Rat Brain in Steriotaxic Coordi- in the rat. Neurosci. Lett. 152:29–32. nates, 2nd ed. Sydney: Academic Press. Li, Y.-Q., M. Takada, and N. Mizuno (1993b) Identification of premotor Popratiloff, A., J.G. Valtschanoff, A. Rustioni, and R.J. Weinberg (1996) interneurons which project bilaterally to the trigeminal motor, facial or Colocalization of GABA and glycine in the rat dorsal column nuclei. hypoglossal nuclei: A fluorescent retrograde double-labeling study in Brain Res. 706:308–312. the rat. Brain Res. 611:160–164. Semba, K., and M.D. Egger (1986) The facial ‘‘motor’’ nerve of the rat: Li, Y.-Q., M. Takada, and N. Mizuno (1993c) The sites of origin of control of vibrissal movement and examination of motor and sensory serotoninergic afferent fibers in the trigeminal motor, facial, and components. J. Comp. Neurol. 247:144–158. hypoglossal nuclei in the rat. Neurosci. Res. 17:307–313. Stein, B.E., R.F. Spencer, and S.B. Edwards (1984) Efferent projections of Li, Y.-Q., M. Takada, T. Kaneko, and N. Mizuno (1996) GABAergic and the neonatal cat superior colliculus: Facial and cerebellum-related glycinergic neurons projecting to the trigeminal motor nucleus: A brainstem structures. J. Comp. Neurol. 230:47–54. double labeling study in the rat. J. Comp. Neurol., 373:498–510. Takada, M., K. Itoh, Y. Yasui, A. Mitani, S. Nomura, and N. Mizuno (1984a) Lowe, A.A. (1981) The neural regulation of tongue movements. Prog. Distribution of premotor neurons for orbicularis oculi motoneurons in Neurobiol. 15:295–344. the cat, with particular reference to possible pathways for blink reflex. Manaker, S., and L.J. Tischler (1993) Origin of serotoninergic afferents to Neurosci. Lett. 50:251–255. the hypoglossal nucleus in the rat. J. Comp. Neurol. 334:466–476. Takada, M., K. Itoh, Y. Yasui, A. Mitani, S. Nomura, and N. Mizuno (1984b) Manaker, S., L.J. Tischler, and A.R. Morrison (1992) Raphespinal and Distribution of premotor neurons for the hypoglossal nucleus in the cat. reticulospinal axon collaterals to the hypoglossal nucleus in the rat. J. Neurosci. Lett. 52:141–146. Comp. Neurol. 322:68–78. Takeuchi, Y., K. Nakano, M. Uemura, K. Matsuda, R. Matsushima, and N. Maxwell, D.J., A.J. Todd, and R. Kerr (1995) Colocalization of glycine and Mizuno (1979) Mesencephalic and pontine afferent fiber system to the GABA in synapses on spinomedullary neurons. Brain Res. 690:127– facial nucleus in the cat: A study using the horseradish peroxidase and 132. silver impregnation techniques. Exp. Neurol. 66:330–342. May, P.L., P.-P. Vidal, and R. Baker (1990) Synaptic organization of Tanaka, T., Y. Takeuchi, and K. Nakano (1978) Cells of origin of the tectal–facial pathways in cat. II. Synaptic potentials following midbrain spino-facial pathway in the cat: A horseradish peroxidase study. Brain tegmentum stimulation. J. Neurophysiol. 64:381–402. Res. 142:580–585. Mizuno, N., S. Nomura, K. Itoh, Y. Nakamura, and A. Konishi (1978) Ter Horst, G.J., J.C.V.M. Copray, R.S.B. Liem, and J.D. Van Willigen (1991) Commissural interneurons for masticatory motoneurons: A light and Projections from the rostral parvocellular reticular formation to pontine electron microscope study using the horseradish peroxidase tracer and medullary nuclei in the rat: involvement in autonomic regulation technique. Exp. Neurol. 59:254–262. and orofacial motor control. Neuroscience 40:735–758. Mizuno, N., Y. Yasui, S. Nomura, K. Itoh, A., Konishi, M. Takada, and M. Todd, A.J., and A.C. Sullivan (1990) Light microscope study of the coexist- Kudo (1983) A light and electron microscopic study of premotor neurons ence of GABA-like and glycine-like immunoreactivities in the spinal for the trigeminal motor nucleus. J. Comp. Neurol. 215:290–298. cord of the rat. J. Comp. Neurol. 296:496–505. Mogoseanu, D., A.D. Smith, and J.P. Bolam (1994) Monosynaptic innerva- Todd, A.J., C. Watt, R.C. Spike, and W. Sieghart (1996) Colocalization of tion of facial motoneurones by neurones of the parvicellular reticular GABA, glycine, and their receptors at synapses in the rat spinal cord. J. formation. Exp. Brain Res. 101:427–438. Neurosci. 16:974–982. Moore, J.K., K.K. Osen, J. Storm-Mathisen, and O.P. Ottersen (1996) Travers, J.B., and L.M. Jackson (1992) Hypoglossal neural activity during g-Aminobutyric acid and glycine in the baboon cochlear nuclei: An licking and swallowing in the awake rat. J. Neurophysiol. 67:1171– immunocytochemical colocalization study with reference to interspecies 1184. differences in inhibitory systems. J. Comp. Neurol. 369:497–519. Travers, J.B., and R. Norgren (1983) Afferent projections to the oral motor Mugnaini, E., and W.H. Oertel (1985) An atlas of the distribution of nuclei in the rat. J. Comp. Neurol. 220:280–298. GABAergic neurons and terminals in the rat CNS as revealed by GAD Tsai, T.C., C.H. Wu, C.Y. Wen, and J.Y. Shieh (1993) Studies of the immunohistochemistry. In A. Bjo¨rklund and T. Ho¨kfelt (eds): Handbook motoneurons following the injection of horseradish peroxidase into the of Chemical Neuroanatomy, vol. 4: GABA and Neuropeptides in the peripheral branches of the facial nerve in rats. Acta Anat. 148:42–48. CNS, part 1. Amsterdam: Elsevier, pp. 436–485. Turman, J.E., Jr., and S.H. Chandler (1994) Immunohistochemical evi- Oertel, W.H., D.E. Schmechel, M.L. Tappaz, and I.J. Kopin (1981) Produc- dence for GABA and glycine-containing trigeminal premotorneurons in tion of a specific antiserum to rat brain glutamic acid decarboxylase by the guinea pig. Synapse 18:7–20. injection of an antigen–antibody complex. Neuroscience 6:2589–2700. Uemura-Sumi, M., M. Itoh, and N. Mizuno (1988) The distribution of Ohta, M., and Y. Moriyama (1986) Supratrigeminal neurons mediate the hypoglossal motoneurons in the dog, rabbit and rat. Anat. Embryol. shortest, disynaptic pathway from the central amygdaloid nucleus to 177:389–394. the contralateral trigeminal motoneurons in the rat. Comp. Biochem. Ugolini, G. (1995) Specificity of rabies virus as a transneuronal tracer of Physiol. 83A:633–641. motor networks: Transfer from hypoglossal motoneurons to connected Ono, T., Y. Ishiwata, N. Inaba, T. Kuroda, and Y. Nakamura (1994) second-order and higher order central nervous system cell groups. J. Hypoglossal premotor neurons with rhythmical inspiratory-related Comp. Neurol. 356:457–480. activity in the cat: Localization and projection to the phrenic nucleus. Watson, C.R.R., S. Sakai, and W. Armstrong (1982) Organization of the Exp. Brain Res. 98:1–12. facial nucleus in the rat. Brain Behav. Evol. 20:19–28. Panneton, W.M., and G.F. Martin (1978) Midbrain projections to the facial Yang, C.C.H., J.Y.H. Chan, and S.H.H. Chan (1995) Excitatory innervation nucleus in the opossum. Brain Res. 145:355–359. of caudal hypoglossal nucleus from nucleus reticularis gigantocellularis Panneton, W.M., and G.F. Martin (1979) Midbrain projections to the in the rat. Neuroscience 65:365–374.