Exp Brain Res (1992) 90:262 270
Exp.erimental BrainResearch 9 Springer-Verlag 1992
Neurons of the motor trigeminal nucleus project to the hypoglossal nucleus in the rat Scott Manaker a, Laura J. Tischler ~, Tracey L. Bigler 1, and Adrian R. Morrison 2 t Pulmonary and Critical Care Division, Department of Medicine, School of Medicine 2 Department of Animal Biology, School of Veterinary Medicine and Center for Sleep and Respiratory Neurobiology, University of Pennsylvania, Philadelphia, PA 19104-4283, USA Received December 10, 1991 / Accepted April 1, 1992
Summary. The hypoglossal nucleus (Mo 12) contains motoneurons that innervate the tongue, while the motor trigeminal nucleus (Mo5) contains motoneurons that elevate or depress the mandible. Previous studies have revealed lateral and medial tegmental field neuronal afferents to the Mo12 adjacent to, but not within, the motor trigeminal nucleus (Mo5). The current studies demonstrate the presence of retrogradely labeled neuronal afferents to the Mo12 within the Mo5 produced by as little as 10 nl of Fast Blue (FB) injected into the Mo12. Retrograde labeling of Mo5 afferents to the Mo12 with injections of Diamidino Yellow (DY) combined with injections of FB into the lumbar spinal cord showed these neuronal afferents to the Mo12 are not part of the diffuse projections to motoneurons from the nucleus subcoeruleus. Retrograde labeling of Mo5 afferents to the Mo12 with DY combined with injections of FB into the masseter revealed these neuronal afferents to the Mo12 are not trigeminal motoneurons. These results in~dicate that Mo5 interneurons are part of the lateral tegmental field projections to the Mo12, and are likely to comprise part of the neural substrate coordinating the motor activity of the jaw and tongue. Key words: Motor trigeminal nucleus - Hypoglossal nucleus Motoneuron - Jaw - Tongue - Rats
Introduction The hypoglossal nucleus (Mo12) contains motoneurons that innervate the tongue and several muscles of the upper airway, such as the genioglossus and geniohyoid (Barnard 1940; Krammer et al. 1979). These motoneurons play a critical role in the physiological functions of the oropharynx, such as mastication, swallowing, and maintaining the patency of the upper airway. Neuronal afferents to the Mo 12 are predominantly localized within Correspondence to: S. Manaker
the lateral tegmental field of the pons and medulla, with smaller numbers of neurons from the medial tegmental field and the nuclei locus coeruleus and subcoeruleus also projecting to the Mo12 (Holstege et al. 1977; Basbaum et al. 1978; Borke et al. 1983; Travers and Norgren 1983; Takada et al. 1984; Ugolini et al. 1987; Aldes 1990). The lateral tegmental field contains propriobulbar interneurons for the Mo12, while the neuronal afferents to the Mo12 from the medial tegmental field and the nuclei locus coeruleus and subcoeruleus are part of the diffuse projections to motoneurons (for review, see Holstege 1991). Like the Mo12, the motor trigeminal nucleus (Mo5) is primarily composed of motoneurons, many of which innervate muscles that elevate or depress the mandible (Mizuno et al. 1975; Limwongse and DeSantis/977; Sasamoto 1979; Yassin and Leong 1979; Jacquin et al. 1983; Lynch/985; Rokx et al. 1985). The Mo5 also contains interneurons (Denavit-Saubier and Corvisier 1972; Sessle 1977; Card et al. 1986) that manifest significant extranuclear projections (Langer etal. 1985; Roste 1989; Ter Horst et al. 1990). However, while many studies have demonstrated that neurons in structures adjacent to the Mo5, such as the intertrigeminal and subcoerulear nuclei, project to the Mo12 (Holstege et al. 1977; Basbaum etal. /978; Borke etal. 1983; Travers and Norgren 1983; Takada et al. 1984; Ugolini et al. 1987; Aldes 1990), almost no data exists to suggest that neurons within the Mo5 project to the Mo12. In the present study, neurons within the Mo5 were labeled by discrete injections of a retrograde fluorescent tracer into the Mo12. To confirm that these neurons were not part of the diffuse projections from the nucleus subcoeruleus to motoneurons, two different retrograde fluorescent tracers were used to compare the distributions of Mo5 neuronal afferents to the Mo12 with the location of pontine neurons projecting to lumbar spinal cord. Within the pontine tegmentum, there was substantial overlap of neurons projecting to the Mo12 with neurons projecting to the lumbar spinal cord, but within the Mo5 only neuronal afferents to the Mo12 were ob-
263 served. Finally, we used two different r e t r o g r a d e tracers. to label t r i g e m i n a l m o t o n e u r o n s p r o j e c t i n g to the masseter a n d to label M o 5 n e u r o n s p r o j e c t i n g to the )Viol2. W h i l e there was overlap o f the two different r e t r o g r a d e l y labeled n e u r o n a l p o p u l a t i o n s w i t h i n the Mo5, n o d o u b l e labeled n e u r o n s were identified, d e m o n s t r a t i n g t h a t the M o 5 n e u r o n a l afferents to the M o 1 2 are a n e u r o n a l p o p u l a t i o n distinct f r o m t r i g e m i n a l m o t o n e u r o n s . We conclude t h a t M o 5 n e u r o n a l afferents to the M o 1 2 are p a r t o f the lateral t e g m e n t a l field p r o p r i o b u l b a r p r o j e c t i o n s to the M o 1 2 c o n s t i t u t i n g the a n a t o m i c basis for p r e v i o u s physiological reports o f i n t e r n e u r o n s w i t h i n the M o 5 ( D e n a v i t - S a u b i e a n d Corvisier 1972; Sessle 1977), a n d are likely to play a n i m p o r t a n t role i n the c o o r d i n a t i o n of jaw and tongue movements.
Methods
Animals Male Sprague-Dawley rats (200-400 g; Charles River Co., Wilmington, Mass., USA) were maintained on a 14/10 light/dark cycle with ad lib food and water, and fasted overnight prior to fluorescent tracer injections.
Surgery and tracer injections All rats received injections of Fast Blue (FB) (3% in distilled water), either alone or in combination with Diamidino Yellow (DY) (3% in distilled water). To retrogradely label neuronal afferents, discrete injections of 10 nl (two cases) or 30 nl (two cases) FB into the Mo12 were performed. In three different cases, DY (2-5 gl, total
Fig. 1A-C. Examples of fluorescent labeling with FB and DY. Thick filled arrow, neuron displaying only nuclear fluorescence from DY; thick empty arrow, neuron displaying only cytoplasmic fluorescence from FB; thin arrow, double labeled neuron displaying both nuclear fluorescence from DY and cytoplasmic fluorescence from FB. A Cytoplasmic fluorescence in a Mo5 neuron retrogradely labeled by 10 nl FB injected into the Mo12. Note the absence of nuclear labeling. B A double labeled neuron with both DY nuclear and FB cytoplasmic fluorescence and a neuron with DY nuclear fluorescence in the nucleus locus coeruleus, following injections of DY into the Mo12 and FB into the lumbar spinal cord. C A trigeminal motoneuron labeled with cytoplasmic fluorescence from FB injected into the masseter, adjacent to a Mo5 neuron
volume) was'injected into the Mo12 and FB (2-5 gl, total volume) was injected into the lumbar spinal cord to determine whether neurons in nuclei and subregions adjacent to the Mo5 were the source of the retrograde labeling from the Mo12. We also wished to determine whether any of the retrogradely labeled cells in the Mo5 were trigeminal motoneurons. To examine this possibility, in another group of three cases DY (2-5 gl, total volume) was injected into the Mo12 and FB (6-20 Ixl, total volume) was injected into the masseter. The masseter was selected for specific study because it is innervated by both large and small motoneurons (Limwongse and DeSantis 1977). For these studies, rats were anesthetized with pentobarbital (75 mg/kg, i.p.), and tracer injections were performed on the right side. For Mo12 injections, the caudal portion of the occipital bone was removed and a micropipette was introduced using the following coordinates: 0.1 mm rostral to the caudal border of the area postrema, 0.3 mm lateral to the midline, and 1.1 mm deep perpendicular to the dorsal medullary surface. For spinal cord injections, a laminectomy was performed at L2 and a micropipette was lowered into the ventral grey matter of the second lumbar segment using the following coordinates: 0.6 mm lateral to the midline and 1.5 mm deep. For injections into the masseter, after the skin was incised the muscle was exposed by blunt dissection and the micropipette placed in the body of the muscle under direct vision. With 40-gm diameter glass micropipettes secured on a 1-gl Hamilton microsyringe, low volume (10 hi) tracer injections of FB were performed over fourteen minutes to prevent any spillage of tracer. Preliminary studies revealed that with this technique, there was no detectable efflux of tracer injectate along the needle path, even with survival intervals of less than 60 min. Large volume (> 1 gl) tracer injections, employed to maximize the detection of double labeled neurons, were performed with 100-gm diameter glass micropipettes secured on a 10-gl Hamilton microsyringe, and occurred over 5 rain. These large tracer volumes were divided into two injections (1.0-2.5 gl) each separated in the rostrocaudal plane by approximately 100 gm for the Mo12 and 300-500 ~tm for the spinal cord. For the masseter, three to eight injections (each 2-5 gl) were made at randomly spaced intervals within the muscle.
displaying only nuclear labeling from DY injected into the Mo12. Again, note the absence of nuclear labeling in the FB labeled neuron; AP, area postrema; cc, central canal; DMN, dorsal motor nucleus of the vagus; DMTg, dorsomedial tegmental nucleus; DR, dorsal raphe; I5, intertrigeminal nucleus; IV, fourth ventricle; LC, locus coeruleus; LPB, lateral parabrachial nucleus; ml, medial lemniscus; Mo5, motor trigeminal nucleus; MPB, medial parabrachial nucleus; NTS, nucleus of the solitary tract; pd, predorsal bundle; P5, principal sensory trigeminal nucleus; PnC, caudal pontine nucleus; PnV, ventral pontine nucleus; py, pyramid; RMg, raphe magnus; RPa, raphe pallidus; RPn, raphe pontis; s5, spinal trigeminal tract; scp, superior cerebellar peduncle; SO, superior olive; SubC, subcoerutear nucleus; XII, hypoglossal nucleus
264 After discrete injection of FB into the Mo12, rats were allowed to survive up to 20 days. After injection of DY into the Mo12 combined with a FB injection into the lumbar spinal cord, rats were allowed to survive for 27-30 days or when combined with a FB injection into the masseter the survival time was 8-12 days.
t i o n sites f o r the f o u r cases r e p o r t e d in this s t u d y rev e a l e d n o e x t e n s i o n o f t r a c e r across the m i d l i n e to the c o n t r a l a t e r a l M o 1 2 , o r to the a d j a c e n t d o r s a l m o t o r nucleus o f the v a g u s o r r e t i c u l a r f o r m a t i o n . Similarly, there was n o evidence o f t r a c e r efflux u p the injection t r a c k in these cases.
Histology After the survival periods described above, rats were anesthetized with pentobarbital (150 mg/kg, i.p.) and transcardially perfused with 100 ml clearing solution (6% Dextran; heparin, 3000 U/l; 20~ followed by 500ml fixative (10% paraformaldehyde; 50 mM sodium phosphate; 20 ~ C) and then 500 ml cryoprotectant (10% sucrose; 100 mM sodium phosphate; 10~ C). The brain and spinal cord were then dissected out, and placed overnight in a second cryoprotectant (30% sucrose; i00 mM sodium phosphate; pH 7.3; 10~ C). Tissues were then blocked, mounted on cryostat chucks and frozen in dry ice. Sequential sections 32 gm thick were cut at - 1 8 ~ C, thaw-mounted on gelatin-chromate coated slides, coverslipped with DPX, and stored with dessicant at - 1 7 ~ C until examination.
Microscopy, photography and data analysis Sections through the pons at the level of the Mo5 were examined under epifluorescence with a Leitz Laborlux 12 microscope, and photography was performed with Kodak T-Max 400. FB fluorescent neurons were defined as only those somata with cytoplasm filled with blue-white fluorescence, a dark non-fluoresceing nucleus, and at least one clearly discernible neuronal process (Fig. 1A, B, C). DY fluorescent neurons were defined as containing the characteristic granular yellow nucleus (Fig. 1 B, C). Neurons doublelabeled, containing both FB and DY, were identified by their filled cytoplasm and brightly fluorescing nuclei (Fig. 1 B) (Kuypers and Huisman 1984). Fluorescent neurons were counted from every third section, except for rats where DY was injected into the Mo12 and FB into the lumbar spinal cord in which case every ninth section was counted. All labeling was manually plotted on camera lucida line drawings derived from adjacent sections stained with cresyl violet. The Mo5 was delineated as the smallest ellipsoid or polygon inclusive of all the large, multipolar e-motoneurons characteristie of the nucleus, bordered ventrally and laterally by the emerging trigeminal motor root fascicles (Mizuno et al. 1975; Limwongse and DeSantis 1977; Sasamoto 1979; Yassin and Leong 1979; Jacquin et al. 1983; Lynch 1985; Rokx et al. 1985; el. Figs. 4A, 6). For tabulation purposes, no distinctions were made between the subdivisions (pars alpha, pars caudalis, pars dorsalis) of the nucleus subcoeruleus, and the labeling within the supratrigeminal nucleus was included with the numerically larger intertrigeminal nucleus. Data are presented as means_+ standard errors.
Results
Localization of Mo5 afferents to the hypoglossal nucleus Injection sites. T h e i n j e c t i o n o f u p to 30 nl F B i n t o the M o 1 2 p r o d u c e d a s m a l l r e g i o n o f necrosis, w i t h s m a l l q u a n t i t i e s o f u n a b s o r b e d , n o n - t r a n s p o r t e d t r a c e r present. S u r r o u n d i n g this c e n t r a l n e c r o t i c r e g i o n were m a n y glia a n d few n e u r o n s , all i n t e n s e l y l a b e l e d w i t h tracer. C i r c u m f e r e n t i a l l y a r o u n d this i n t e r m e d i a t e r e g i o n was a r e g i o n o f h e a v i l y l a b e l e d n e u r o n s , w i t h few glia identified, all c o n t a i n e d w i t h i n the limits o f the r i g h t M o 1 2 (Fig. 2). T h e M o 1 2 i n j e c t i o n sites were a l w a y s c o n t a i n e d w i t h i n the r o s t r o c a u d a l e x t e n t o f the nucleus. T h e injec-
Retrograde labeling. I n general, the m a j o r i t y o f n e u r o n a l afferents to the M o 1 2 f r o m the m e d u l l a a n d c a u d a l p o n s in all f o u r cases were l o c a t e d w i t h i n t h e l a t e r a l t e g m e n t a l field, a n d the r e m a i n d e r were o b s e r v e d w i t h i n the m e d i a l t e g m e n t a l field. These findings were l a r g e l y c o n f i r m a t o r y o f p r e v i o u s studies o f the afferent c o n n e c t i v i t y to the M o 12 ( H o l s t e g e et aL 1977; B a s b a u m et al. 1978; B o r k e e t a l . 1983; T r a v e r s a n d N o r g r e n 1983; T a k a d a et al. 1984; U g o l i n i et al. 1987; A l d e s 1990), a n d for b r e v i t y will t h e r e f o r e n o t be f u r t h e r d e s c r i b e d . W i t h i n the M o 5 , we o b s e r v e d a m o d e r a t e n u m b e r o f c i r c u m f e r e n t i a l l y d i s t r i b u t e d n e u r o n s r e t r o g r a d e l y lab e l e d w i t h F B (Fig. 3). T h e n u m b e r o f F B l a b e l e d neur o n s w i t h i n the M o 5 i p s i l a t e r a l ( 1 2 5 + 1 9 n e u r o n s ) to the h y p o g l o s s a l i n j e c t i o n site was g r e a t e r t h a n t h a t o b served c o n t r a l a t e r a l l y ( 3 5 + 7 n e u r o n s ) in each o f the f o u r cases, a n d the n e u r o n s were p r e s e n t t h r o u g h o u t the r o s t r o c a u d a l e x t e n t o f the nucleus. T h e s e n e u r o n s were s m a l l e r in size t h a n w h a t a p p e a r e d to be the large, trigeminal motoneurons. The majority of Mo5 neuronal afferents to the M o 1 2 were l o c a t e d a t the p e r i p h e r y o f the nucleus, s o m e t i m e s i n t e r s p e r s e d a m o n g w h a t a p p e a r e d to be t r i g e m i n a l m o t o n e u r o n s , a n d less c o m m o n ly o b s e r v e d in the c e n t e r o f the nucleus. W h i l e o v e r h a l f o f the n e u r o n s r e t r o g r a d e l y l a b e l e d w i t h F B were n o t e d a l o n g the v e n t r a l a n d l a t e r a l b o r d e r s o f the nucleus, subs t a n t i a l n u m b e r s were also o b s e r v e d a l o n g the m e d i a l a n d d o r s a l edges o f the nucleus. I n the a d j a c e n t i n t e r t r i g e m i n a l nucleus, m o d e r a t e numbers of retrogradely labeled neurons werepresent >
Fig. 2A-H. Example of a Mo12 injection site after microinjection of 30 nl FB. A, B, C and D are fluorescent microphotographs showing the extent of the injection site. B is the center of the injection; while A, C and D are 100 Ixm caudal, 200 gm rostral and 300 gm rostral to B, respectively. For comparison, F and G are bright field microphotographs of the same tissue sections shown in B and C, respectively, after counterstaining with cresyl violet. The arrowheads in these four panels (B, C, F, G) point to the central region of necrosis, within which some residual, unabsorbed tracer may be discerned. For orientation, E is a low-power bright field microphotograph of the same tissue sections shown in B and F; and reveals this region to be caudal medulla, just caudal to the obex. Similarly, H is a lowpower bright-field microphotograph of the same tissue section shown in C and G; and reveals this region to be caudal medulla rostral to the obex. The broken lines in E and H outline the magnified regions of F and G, respectively, Magnification bar in A: 500 gm for E and H; 200 gm for all other panels. Note that the central necrosis, the surrounding area of glial reaction and unabsorbed tracer, and the circumferential region of very brightly fluorescing neurons which comprise the injection site are contained entirely within the Mo12; and do not extend into the dorsal motor nucleus of the vagus, into the adjacent reticular formation, or across the midline. The entire rostrocaudal extent of the injection site was also contained within the boundaries of the Mo12; for abbreviations see Fig. I
265
Fig. 2.
266
\\
I
, '
/
\\ ~i \
,
,' I
< ~
/
',: :. ~
-
-,
~ ) y/
-;:.;-'
Exp.erimental BrainResearch 9 Springer-Verlag 1992
Neurons of the motor trigeminal nucleus project to the hypoglossal nucleus in the rat Scott Manaker a, Laura J. Tischler ~, Tracey L. Bigler 1, and Adrian R. Morrison 2 t Pulmonary and Critical Care Division, Department of Medicine, School of Medicine 2 Department of Animal Biology, School of Veterinary Medicine and Center for Sleep and Respiratory Neurobiology, University of Pennsylvania, Philadelphia, PA 19104-4283, USA Received December 10, 1991 / Accepted April 1, 1992
Summary. The hypoglossal nucleus (Mo 12) contains motoneurons that innervate the tongue, while the motor trigeminal nucleus (Mo5) contains motoneurons that elevate or depress the mandible. Previous studies have revealed lateral and medial tegmental field neuronal afferents to the Mo12 adjacent to, but not within, the motor trigeminal nucleus (Mo5). The current studies demonstrate the presence of retrogradely labeled neuronal afferents to the Mo12 within the Mo5 produced by as little as 10 nl of Fast Blue (FB) injected into the Mo12. Retrograde labeling of Mo5 afferents to the Mo12 with injections of Diamidino Yellow (DY) combined with injections of FB into the lumbar spinal cord showed these neuronal afferents to the Mo12 are not part of the diffuse projections to motoneurons from the nucleus subcoeruleus. Retrograde labeling of Mo5 afferents to the Mo12 with DY combined with injections of FB into the masseter revealed these neuronal afferents to the Mo12 are not trigeminal motoneurons. These results in~dicate that Mo5 interneurons are part of the lateral tegmental field projections to the Mo12, and are likely to comprise part of the neural substrate coordinating the motor activity of the jaw and tongue. Key words: Motor trigeminal nucleus - Hypoglossal nucleus Motoneuron - Jaw - Tongue - Rats
Introduction The hypoglossal nucleus (Mo12) contains motoneurons that innervate the tongue and several muscles of the upper airway, such as the genioglossus and geniohyoid (Barnard 1940; Krammer et al. 1979). These motoneurons play a critical role in the physiological functions of the oropharynx, such as mastication, swallowing, and maintaining the patency of the upper airway. Neuronal afferents to the Mo 12 are predominantly localized within Correspondence to: S. Manaker
the lateral tegmental field of the pons and medulla, with smaller numbers of neurons from the medial tegmental field and the nuclei locus coeruleus and subcoeruleus also projecting to the Mo12 (Holstege et al. 1977; Basbaum et al. 1978; Borke et al. 1983; Travers and Norgren 1983; Takada et al. 1984; Ugolini et al. 1987; Aldes 1990). The lateral tegmental field contains propriobulbar interneurons for the Mo12, while the neuronal afferents to the Mo12 from the medial tegmental field and the nuclei locus coeruleus and subcoeruleus are part of the diffuse projections to motoneurons (for review, see Holstege 1991). Like the Mo12, the motor trigeminal nucleus (Mo5) is primarily composed of motoneurons, many of which innervate muscles that elevate or depress the mandible (Mizuno et al. 1975; Limwongse and DeSantis/977; Sasamoto 1979; Yassin and Leong 1979; Jacquin et al. 1983; Lynch/985; Rokx et al. 1985). The Mo5 also contains interneurons (Denavit-Saubier and Corvisier 1972; Sessle 1977; Card et al. 1986) that manifest significant extranuclear projections (Langer etal. 1985; Roste 1989; Ter Horst et al. 1990). However, while many studies have demonstrated that neurons in structures adjacent to the Mo5, such as the intertrigeminal and subcoerulear nuclei, project to the Mo12 (Holstege et al. 1977; Basbaum etal. /978; Borke etal. 1983; Travers and Norgren 1983; Takada et al. 1984; Ugolini et al. 1987; Aldes 1990), almost no data exists to suggest that neurons within the Mo5 project to the Mo12. In the present study, neurons within the Mo5 were labeled by discrete injections of a retrograde fluorescent tracer into the Mo12. To confirm that these neurons were not part of the diffuse projections from the nucleus subcoeruleus to motoneurons, two different retrograde fluorescent tracers were used to compare the distributions of Mo5 neuronal afferents to the Mo12 with the location of pontine neurons projecting to lumbar spinal cord. Within the pontine tegmentum, there was substantial overlap of neurons projecting to the Mo12 with neurons projecting to the lumbar spinal cord, but within the Mo5 only neuronal afferents to the Mo12 were ob-
263 served. Finally, we used two different r e t r o g r a d e tracers. to label t r i g e m i n a l m o t o n e u r o n s p r o j e c t i n g to the masseter a n d to label M o 5 n e u r o n s p r o j e c t i n g to the )Viol2. W h i l e there was overlap o f the two different r e t r o g r a d e l y labeled n e u r o n a l p o p u l a t i o n s w i t h i n the Mo5, n o d o u b l e labeled n e u r o n s were identified, d e m o n s t r a t i n g t h a t the M o 5 n e u r o n a l afferents to the M o 1 2 are a n e u r o n a l p o p u l a t i o n distinct f r o m t r i g e m i n a l m o t o n e u r o n s . We conclude t h a t M o 5 n e u r o n a l afferents to the M o 1 2 are p a r t o f the lateral t e g m e n t a l field p r o p r i o b u l b a r p r o j e c t i o n s to the M o 1 2 c o n s t i t u t i n g the a n a t o m i c basis for p r e v i o u s physiological reports o f i n t e r n e u r o n s w i t h i n the M o 5 ( D e n a v i t - S a u b i e a n d Corvisier 1972; Sessle 1977), a n d are likely to play a n i m p o r t a n t role i n the c o o r d i n a t i o n of jaw and tongue movements.
Methods
Animals Male Sprague-Dawley rats (200-400 g; Charles River Co., Wilmington, Mass., USA) were maintained on a 14/10 light/dark cycle with ad lib food and water, and fasted overnight prior to fluorescent tracer injections.
Surgery and tracer injections All rats received injections of Fast Blue (FB) (3% in distilled water), either alone or in combination with Diamidino Yellow (DY) (3% in distilled water). To retrogradely label neuronal afferents, discrete injections of 10 nl (two cases) or 30 nl (two cases) FB into the Mo12 were performed. In three different cases, DY (2-5 gl, total
Fig. 1A-C. Examples of fluorescent labeling with FB and DY. Thick filled arrow, neuron displaying only nuclear fluorescence from DY; thick empty arrow, neuron displaying only cytoplasmic fluorescence from FB; thin arrow, double labeled neuron displaying both nuclear fluorescence from DY and cytoplasmic fluorescence from FB. A Cytoplasmic fluorescence in a Mo5 neuron retrogradely labeled by 10 nl FB injected into the Mo12. Note the absence of nuclear labeling. B A double labeled neuron with both DY nuclear and FB cytoplasmic fluorescence and a neuron with DY nuclear fluorescence in the nucleus locus coeruleus, following injections of DY into the Mo12 and FB into the lumbar spinal cord. C A trigeminal motoneuron labeled with cytoplasmic fluorescence from FB injected into the masseter, adjacent to a Mo5 neuron
volume) was'injected into the Mo12 and FB (2-5 gl, total volume) was injected into the lumbar spinal cord to determine whether neurons in nuclei and subregions adjacent to the Mo5 were the source of the retrograde labeling from the Mo12. We also wished to determine whether any of the retrogradely labeled cells in the Mo5 were trigeminal motoneurons. To examine this possibility, in another group of three cases DY (2-5 gl, total volume) was injected into the Mo12 and FB (6-20 Ixl, total volume) was injected into the masseter. The masseter was selected for specific study because it is innervated by both large and small motoneurons (Limwongse and DeSantis 1977). For these studies, rats were anesthetized with pentobarbital (75 mg/kg, i.p.), and tracer injections were performed on the right side. For Mo12 injections, the caudal portion of the occipital bone was removed and a micropipette was introduced using the following coordinates: 0.1 mm rostral to the caudal border of the area postrema, 0.3 mm lateral to the midline, and 1.1 mm deep perpendicular to the dorsal medullary surface. For spinal cord injections, a laminectomy was performed at L2 and a micropipette was lowered into the ventral grey matter of the second lumbar segment using the following coordinates: 0.6 mm lateral to the midline and 1.5 mm deep. For injections into the masseter, after the skin was incised the muscle was exposed by blunt dissection and the micropipette placed in the body of the muscle under direct vision. With 40-gm diameter glass micropipettes secured on a 1-gl Hamilton microsyringe, low volume (10 hi) tracer injections of FB were performed over fourteen minutes to prevent any spillage of tracer. Preliminary studies revealed that with this technique, there was no detectable efflux of tracer injectate along the needle path, even with survival intervals of less than 60 min. Large volume (> 1 gl) tracer injections, employed to maximize the detection of double labeled neurons, were performed with 100-gm diameter glass micropipettes secured on a 10-gl Hamilton microsyringe, and occurred over 5 rain. These large tracer volumes were divided into two injections (1.0-2.5 gl) each separated in the rostrocaudal plane by approximately 100 gm for the Mo12 and 300-500 ~tm for the spinal cord. For the masseter, three to eight injections (each 2-5 gl) were made at randomly spaced intervals within the muscle.
displaying only nuclear labeling from DY injected into the Mo12. Again, note the absence of nuclear labeling in the FB labeled neuron; AP, area postrema; cc, central canal; DMN, dorsal motor nucleus of the vagus; DMTg, dorsomedial tegmental nucleus; DR, dorsal raphe; I5, intertrigeminal nucleus; IV, fourth ventricle; LC, locus coeruleus; LPB, lateral parabrachial nucleus; ml, medial lemniscus; Mo5, motor trigeminal nucleus; MPB, medial parabrachial nucleus; NTS, nucleus of the solitary tract; pd, predorsal bundle; P5, principal sensory trigeminal nucleus; PnC, caudal pontine nucleus; PnV, ventral pontine nucleus; py, pyramid; RMg, raphe magnus; RPa, raphe pallidus; RPn, raphe pontis; s5, spinal trigeminal tract; scp, superior cerebellar peduncle; SO, superior olive; SubC, subcoerutear nucleus; XII, hypoglossal nucleus
264 After discrete injection of FB into the Mo12, rats were allowed to survive up to 20 days. After injection of DY into the Mo12 combined with a FB injection into the lumbar spinal cord, rats were allowed to survive for 27-30 days or when combined with a FB injection into the masseter the survival time was 8-12 days.
t i o n sites f o r the f o u r cases r e p o r t e d in this s t u d y rev e a l e d n o e x t e n s i o n o f t r a c e r across the m i d l i n e to the c o n t r a l a t e r a l M o 1 2 , o r to the a d j a c e n t d o r s a l m o t o r nucleus o f the v a g u s o r r e t i c u l a r f o r m a t i o n . Similarly, there was n o evidence o f t r a c e r efflux u p the injection t r a c k in these cases.
Histology After the survival periods described above, rats were anesthetized with pentobarbital (150 mg/kg, i.p.) and transcardially perfused with 100 ml clearing solution (6% Dextran; heparin, 3000 U/l; 20~ followed by 500ml fixative (10% paraformaldehyde; 50 mM sodium phosphate; 20 ~ C) and then 500 ml cryoprotectant (10% sucrose; 100 mM sodium phosphate; 10~ C). The brain and spinal cord were then dissected out, and placed overnight in a second cryoprotectant (30% sucrose; i00 mM sodium phosphate; pH 7.3; 10~ C). Tissues were then blocked, mounted on cryostat chucks and frozen in dry ice. Sequential sections 32 gm thick were cut at - 1 8 ~ C, thaw-mounted on gelatin-chromate coated slides, coverslipped with DPX, and stored with dessicant at - 1 7 ~ C until examination.
Microscopy, photography and data analysis Sections through the pons at the level of the Mo5 were examined under epifluorescence with a Leitz Laborlux 12 microscope, and photography was performed with Kodak T-Max 400. FB fluorescent neurons were defined as only those somata with cytoplasm filled with blue-white fluorescence, a dark non-fluoresceing nucleus, and at least one clearly discernible neuronal process (Fig. 1A, B, C). DY fluorescent neurons were defined as containing the characteristic granular yellow nucleus (Fig. 1 B, C). Neurons doublelabeled, containing both FB and DY, were identified by their filled cytoplasm and brightly fluorescing nuclei (Fig. 1 B) (Kuypers and Huisman 1984). Fluorescent neurons were counted from every third section, except for rats where DY was injected into the Mo12 and FB into the lumbar spinal cord in which case every ninth section was counted. All labeling was manually plotted on camera lucida line drawings derived from adjacent sections stained with cresyl violet. The Mo5 was delineated as the smallest ellipsoid or polygon inclusive of all the large, multipolar e-motoneurons characteristie of the nucleus, bordered ventrally and laterally by the emerging trigeminal motor root fascicles (Mizuno et al. 1975; Limwongse and DeSantis 1977; Sasamoto 1979; Yassin and Leong 1979; Jacquin et al. 1983; Lynch 1985; Rokx et al. 1985; el. Figs. 4A, 6). For tabulation purposes, no distinctions were made between the subdivisions (pars alpha, pars caudalis, pars dorsalis) of the nucleus subcoeruleus, and the labeling within the supratrigeminal nucleus was included with the numerically larger intertrigeminal nucleus. Data are presented as means_+ standard errors.
Results
Localization of Mo5 afferents to the hypoglossal nucleus Injection sites. T h e i n j e c t i o n o f u p to 30 nl F B i n t o the M o 1 2 p r o d u c e d a s m a l l r e g i o n o f necrosis, w i t h s m a l l q u a n t i t i e s o f u n a b s o r b e d , n o n - t r a n s p o r t e d t r a c e r present. S u r r o u n d i n g this c e n t r a l n e c r o t i c r e g i o n were m a n y glia a n d few n e u r o n s , all i n t e n s e l y l a b e l e d w i t h tracer. C i r c u m f e r e n t i a l l y a r o u n d this i n t e r m e d i a t e r e g i o n was a r e g i o n o f h e a v i l y l a b e l e d n e u r o n s , w i t h few glia identified, all c o n t a i n e d w i t h i n the limits o f the r i g h t M o 1 2 (Fig. 2). T h e M o 1 2 i n j e c t i o n sites were a l w a y s c o n t a i n e d w i t h i n the r o s t r o c a u d a l e x t e n t o f the nucleus. T h e injec-
Retrograde labeling. I n general, the m a j o r i t y o f n e u r o n a l afferents to the M o 1 2 f r o m the m e d u l l a a n d c a u d a l p o n s in all f o u r cases were l o c a t e d w i t h i n t h e l a t e r a l t e g m e n t a l field, a n d the r e m a i n d e r were o b s e r v e d w i t h i n the m e d i a l t e g m e n t a l field. These findings were l a r g e l y c o n f i r m a t o r y o f p r e v i o u s studies o f the afferent c o n n e c t i v i t y to the M o 12 ( H o l s t e g e et aL 1977; B a s b a u m et al. 1978; B o r k e e t a l . 1983; T r a v e r s a n d N o r g r e n 1983; T a k a d a et al. 1984; U g o l i n i et al. 1987; A l d e s 1990), a n d for b r e v i t y will t h e r e f o r e n o t be f u r t h e r d e s c r i b e d . W i t h i n the M o 5 , we o b s e r v e d a m o d e r a t e n u m b e r o f c i r c u m f e r e n t i a l l y d i s t r i b u t e d n e u r o n s r e t r o g r a d e l y lab e l e d w i t h F B (Fig. 3). T h e n u m b e r o f F B l a b e l e d neur o n s w i t h i n the M o 5 i p s i l a t e r a l ( 1 2 5 + 1 9 n e u r o n s ) to the h y p o g l o s s a l i n j e c t i o n site was g r e a t e r t h a n t h a t o b served c o n t r a l a t e r a l l y ( 3 5 + 7 n e u r o n s ) in each o f the f o u r cases, a n d the n e u r o n s were p r e s e n t t h r o u g h o u t the r o s t r o c a u d a l e x t e n t o f the nucleus. T h e s e n e u r o n s were s m a l l e r in size t h a n w h a t a p p e a r e d to be the large, trigeminal motoneurons. The majority of Mo5 neuronal afferents to the M o 1 2 were l o c a t e d a t the p e r i p h e r y o f the nucleus, s o m e t i m e s i n t e r s p e r s e d a m o n g w h a t a p p e a r e d to be t r i g e m i n a l m o t o n e u r o n s , a n d less c o m m o n ly o b s e r v e d in the c e n t e r o f the nucleus. W h i l e o v e r h a l f o f the n e u r o n s r e t r o g r a d e l y l a b e l e d w i t h F B were n o t e d a l o n g the v e n t r a l a n d l a t e r a l b o r d e r s o f the nucleus, subs t a n t i a l n u m b e r s were also o b s e r v e d a l o n g the m e d i a l a n d d o r s a l edges o f the nucleus. I n the a d j a c e n t i n t e r t r i g e m i n a l nucleus, m o d e r a t e numbers of retrogradely labeled neurons werepresent >
Fig. 2A-H. Example of a Mo12 injection site after microinjection of 30 nl FB. A, B, C and D are fluorescent microphotographs showing the extent of the injection site. B is the center of the injection; while A, C and D are 100 Ixm caudal, 200 gm rostral and 300 gm rostral to B, respectively. For comparison, F and G are bright field microphotographs of the same tissue sections shown in B and C, respectively, after counterstaining with cresyl violet. The arrowheads in these four panels (B, C, F, G) point to the central region of necrosis, within which some residual, unabsorbed tracer may be discerned. For orientation, E is a low-power bright field microphotograph of the same tissue sections shown in B and F; and reveals this region to be caudal medulla, just caudal to the obex. Similarly, H is a lowpower bright-field microphotograph of the same tissue section shown in C and G; and reveals this region to be caudal medulla rostral to the obex. The broken lines in E and H outline the magnified regions of F and G, respectively, Magnification bar in A: 500 gm for E and H; 200 gm for all other panels. Note that the central necrosis, the surrounding area of glial reaction and unabsorbed tracer, and the circumferential region of very brightly fluorescing neurons which comprise the injection site are contained entirely within the Mo12; and do not extend into the dorsal motor nucleus of the vagus, into the adjacent reticular formation, or across the midline. The entire rostrocaudal extent of the injection site was also contained within the boundaries of the Mo12; for abbreviations see Fig. I
265
Fig. 2.
266
\\
I
, '
/
\\ ~i \
,
,' I
< ~
/
',: :. ~
-
-,
~ ) y/
-;:.;-'
