Exp Brain Res (1990) 81 : 167-178

Experimental BrainResearch 9 Springer-Verlag1990

Catecholamine-containing axon terminals in the hypoglossal nucleus of the rat: an immuno-electronmicroscopic study L.D. Aides 1, B. Shaw 1, R. B. Chronister 1' 2, and J.W. Haycock 3 1 Department of Structural and Cellular Biologyand 2 Department of Neurology, University of South Alabama, College of Medicine, Mobile, AL 36688, USA a Department of Biochemistryand Molecular Biology,Louisiana State University, College of Medicine, New Orleans, LA, USA Received August 7, 1989 / Accepted January 22, 1990

Summary. A correlative light and electron microscopic investigation was undertaken to determine the morphology and distribution of catecholamine (CA)-containing axon terminals in the hypoglossal nucleus (XII) of the rat. This was accomplished immunocytochemically with antibody to tyrosine hydroxylase (TH). The major findings in this study were the following: 1) Immunoreactive profiles were found throughout XII and included unmyelinated axons, varicosities, axon terminals and dendrites; 2) Nonsynaptic immunoreactive profiles (preterminal axons, varicosities) were more frequently observed (55.2%) than synaptic profiles (43.5%); 3) CA-containing axon terminals ending on dendrites were more numerous (71.8%) than those synapsing on somata (25.4%) or nonlabeled axon terminals (2.7%); 4) The morphology of labeled axon terminals was variable. Axodendritic terminals typically contained numerous small, round agranular vesicles, a few large dense-core vesicles and were associated with either a symmetric or no synaptic specialization, axosomatic terminals were often associated with a presynaptic membrane thickening or a symmetric synaptic specialization and contained small, round and a few elliptical-shaped vesicles, while axoaxonic synapses formed asymmetric postsynaptic specializations; and 5) CA-positive dendritic processes were identified in XII. These findings confirm the CA innervation of XII, and suggest a complex, multifunctional role for CA in controlling oro-lingual motor behavior. Key words: Hypoglossal nucleus - Catecholamines Norepinephrine - Immunocytochemistry - Electron microscopy - Rat

Introduction Results obtained over the past decade have established that the hypoglossal nucleus (XII) receives a moderately Offprint requests to: L.D. Aldes (address see above)

dense catecholamine (CA) innervation (Swanson and Hartman 1975; Levitt and Moore 1979; Palkovits and Jacobowits 1979; Kalia et al. 1985; Aldes et al. 1988a, c). Recent studies have further characterized this input by demonstrating that a specific subgrouping of protrusor motoneurons in the caudoventromedial quadrant of XII is the principal target CA afferents (Aldes et al. 1988a, c), and that the CA innervation of XII is largely, if not exclusively, noradrenergic (NE - Levitt and Moore 1979; Aldes et al. 1988c). These observations are of particular interest since (1) tongue protrusor activity is important in maintaining the patency of the upper airway (Sauerland and Mitchell 1970, 1975; Remmers et al. 1978; Brouillette and Thach 1979, 1980), in deglutition, mastication, suckling and, in humans, articulated speech (see Dubner et al. 1978; Lowe 1981); (2) NE is involved in modulating spinal motoneuron activity and reflexes (Strahlendorf et al. 1980; White and Neuman 1980, 1983; Fung and Barnes 1981; Chan et al. 1986; Wohlberg et al. 1986) and a similar function for NE is likely in XII; and (3) different neurotransmitter/neuromodulator systems (Aldes et al. 1988a-c) and brainstem afferents (Ermini and Aldes 1988; Aldes and Chapman, in preparation; see Note added in proof) terminate predominantly in the same protrusor motoneuron subgrouping as NE as do other neural systems (Elmund et al. 1983; Mameli and Tolu 1985, 1986). In order to understand the precise role of NE in tongue control, and tongue protrusor activity in particular, it is necessary to elucidate the synaptic relationships that exist between CA axon terminals and XII motoneurons. To our knowledge this has not been investigated in any species, although the NE innervation of other lower motoneuron groups has been described (Mizukawa and Takeuchi 1982; Akeyson et al. 1983; Kojima et al. 1985; Card et al. 1986). In the present study we sought to characterize the morphology of CA axon terminals in XII and to determine on what postsynaptic structures they terminate. This was accomplished immunocytochemicallywith anti-

168 b o d y to t y r o s i n e h y d r o x y l a s e ( T H - H a y c o c k 1987; A l d e s et al. 1988c), the r a t e limiting e n z y m e in the b i o s y n thesis o f C A ( N a g a t s u et al. 1964; L e v i t t et al. 1965).

Methods

Immunoc y t o chemistr y Nine female, Sprague-Dawley rats (175-200 g) were used in this study. After anesthetization with ketamine hydrochloride (50 mg/ kg; IM) and acepromazine maleate (0.05 mg/kg; IM), animals were perfused-fixed transcardially with 4% paraformaldehyde, 0.25% glutaraldehyde (TABB) and 1.0 % sucrose in 0.1 M phosphate buffer, pH 7.4. Brains were removed from the cranial vault, blocked, postfixed in 4% paraformaldehyde for 2-4 h and rinsed in buffer containing 5% sucrose overnight at 4 ~ C. Coronal sections (30-50 gin) were cut through XII on a Vibratome (Oxford) into phosphatebuffered-saline (PBS; pH 7.4) and rinsed (3 x 10 min). Sections were placed in a blocking solution containing 1% bovine-serum-albumin (BSA - Sigma; St. Louis, MO), 10% goat serum (GS - Tago; Burlingame, CA), 0.1% Triton-X 100 detergent in 0.1 M PBS (pH - 7.4) for 30 min at 22 ~ C to diminish non-specific staining. Sections were then incubated in rabbit anti-TH (Haycock 1987; Aldes et al. 1988c) at a dilution of 1 : 10 000 in carrier (0.1 M PBS, pH 7.4, 1% BSA, 1% GS, 0.1% Triton-X 100) for 36--48 h at 4~ C. Next, sections were rinsed in carrier without Triton-X 100 and incubated in biotinylated goat-anti rabbit IgG (Pc1 Freez; Brown Deer, WI) at a concentration of 1 : 200 for 30 min at 22 ~ C. After rinsing several times in carrier, sections were transferred to Avidin-Biotin Complex (ABC; Pel Freez, Deer Park, WI; Hsu et al. 1981) prepared in PBS containing 0.1% Tween 20 (1 : 250) for 1 h at 22 ~ C. Sections were rinsed thoroughly in PBS, reacted for 3-5 min in cold DAB (0.02% in 0.0007% H202) and rinsed again in PBS (3 x 10 min).

E M preparation Sections were transferred to 0.1 M phosphate buffer (PB-pH 7.4), osmicated (1-2%, pH 7.4) at room temperature for 1-2 h and washed several times in PB for 30-60 rain. Next, sections were rinsed briefly in double-distilled water, dehydrated in alcohol, then acetone, infiltrated in Spurr resin and flat-embedded between silanized glass microscope slides (Aides and Boone 1984). Selected areas of XII were identified in the light microscope and photographed. After placing a small etch-mark in the plastic overlying the area of interest, thin sections were cut on an ultramicrotome (LKB), placed on copper or nickel 300 mesh grids, counterstained (Reynolds lead citrate) and examined in a transmission electron microscope (Philips 301).

Table 1. Number and distribution of labeled CA profiles in XII. Nonsynaptic - preterminal axons and varicosities; Synaptic - axodendritic, axosomatic and axoaxonic contacts; Other - labeled dendrites (N = 6) Nonsynaptic Synaptic Other

279 220 6

Total

505

(55.2 %) (43.5 %) (1.1%)

Table 2. Number and distribution of labeled synaptic profiles in XII

Axodendritic Axosomatic Axoaxonic

158 56 6

Total

220

% synaptic

% total

71.8 % 25.4% 2.7 %

31.2 % 11.0% 1.1%

descriptions were based on observations of over 800 labeled profiles that included samples from each animal and from all regions of XII. Two categories of labeled profiles were distinguished: nonsynaptic and synaptic. Structures included in the former category included preterminal axons and varicosities. These profiles made no direct synaptic contact with any postsynaptic element. Conversely, labeled profiles were defined as synaptic if there was close apposition of preand postsynaptic membranes without an intervening glial membrane and by the presence of synaptic vesicles and synaptic cleft with or without membrane specialization. Quantitative analyses were performed on 24 grids from 4 animals. Grids were selected on the basis of intensity of immunoreactivity, number of labeled profiles and adequacy of ultrastructure (Aides et al. 1989). Each grid contained sections through the first few microns of tissue. All together, approximately 450 000 gm 2 of tissue was analyzed (10 sections/grid; 2025 gmZ/grid square; 24 grids). Although sections from all regions of XII were analyzed, owing to the greater density of immunoreactivity in the caudoventromedial quadrant (Fig. 1A), the majority of grids (N= 16) contained sections from this area. This procedure ensured an adequate sample size. Counts were made of all labeled profiles on each grid square and measurements were made from negatives. Data from each grid were added together since no differences were seen in the morphology of labeled terminals or the distribution of labeled terminals on dendrites versus somata in different regions of XII. A total of 505 labeled profiles form the basis of our quantitative results (Tables 1 and 2).

Controls Results

Control sections through XII, randomly selected from each animal, were incubated without primary antiserum either in carrier alone or in carrier saturated (20%) with rabbit serum. All other steps were identical to those conducted for non-control sections. Comparisons were made between control sections and those incubated with antiTH for the presence ofimmunoreactivity (Figs. 1A, B). The specificity and cross-reactivity of the TH antibody have been previously described (Haycock 1987).

Data analysis Sections through rostral, middle and caudal regions of XII were examined for labeled TH profiles (Aldes et al. 1988a, 1989). Labeled profiles were identified by the presence of electron dense immunoprecipitate that was not seen in control sections. Qualitative

L i g h t m i c r o s c o p i c e v a l u a t i o n o f f l a t - e m b e d d e d sections i n c u b a t e d in T H a n t i b o d y r e v e a l e d i m m u n o r e a c t i v e profiles in all r e g i o n s o f X I I . T h e c a u d o v e n t r o m e d i a l q u a d r a n t c o n t a i n e d the g r e a t e s t d e n s i t y o f i m m u n o r e a c tivity (Fig. 1A). T H - p o s i t i v e p r o c e s s e s were m a i n l y o f small d i a m e t e r (0.2-0.75 g m ) a n d a s s o c i a t e d w i t h varicosities (0.5-2.0 g m in d i a m e t e r ) , a l t h o u g h l a r g e r processes (1.0-2.4 ~tm) d e v o i d o f varicosities also were o b s e r v e d (Fig. 1A, B). Profiles s u r r o u n d i n g m o t o n e u r o n s (Fig. 1B) were seen t h r o u g h o u t X I I a n d were especially pronounced caudoventromedially. A t the e l e c t r o n m i c r o s c o p i c level, i m m u n o r e a c t i v e profiles p o s i t i v e for T H were o b s e r v e d in all r e g i o n s o f

169

Fig. 1. A Light microscopic photomicrograph of a plastic (flatembedded) section demonstrating the distribution pattern of TH immunoreactivity in the caudal third of the hypoglossal nucleus (XII). Note the focal innervation of the ventromedial quadrant (*). The intense staining in the dorsal vagal nucleus is indicated (arrows). Scale bar - 0.15 ram. B The perisomatic distribution of THimmunoreactive profiles (black, elongate fibers and punctate dots)

around XII motoneurons is demonstrated. The unstained nucleoli of several motoneurons are visible. Scale bar - 25 ~tm. C Photomicrograph of a flat-embedded control section incubated without primary antibody showing no immunoreactivity in XII or the dorsal vagal (X)/solitarius nuclei (compare with Fig. 1A). Darkened fibers (arrow) are myelinated axons of XII motoneurons stained by osmium during EM fixation process. Scale bar - 0.25 mm

XII. Labeled profiles included u n m y e l i n a t e d axons, varicosities, a x o n terminals and occasionally dendrites (Fig. 2). U n m y e l i n a t e d axons (Figs. 2 A ; 3A) a n d varicosities (Figs. 2 A ; 3B) were the m o s t frequently observed labeled profiles in a n y section and a c c o u n t e d for 55.2% o f the total n u m b e r o f labeled profiles identified in this area

(Table 1). U n m y e l i n a t e d axons averaged 0.16 g m in diameter and typically c o n t a i n e d microtubules (Figs. 3A). Labeled preterminal axons were often seen coursing t o w a r d and a r o u n d unlabeled dendrites (not shown). Varicosities r a n g e d in size f r o m 0.22-0.74 gm, but in contrast to u n m y e l i n a t e d axons, contained variable n u m -

170

Fig. 2. A Electron micrograph of XII showing a long TH-positive preterminal axon (small arrowheads) with electron dense immunoprecipitate, small (clear arrow) and large (black arrow) varicosities, and a labeled dendrite (curved arrow). Scale bar - 0.48 gm. B Electron micrograph through a dendritic field in XII showing

two TH-positive terminals (black arrows) synapsing on separate medium sized dendrites (D). Non-labeled axon terminals (clear arrows) synapsing on other dendrites are indicated. Scale bar 0.73 [am

Fig. 3. A A TH-positive preterminal axon (arrow) among several non-labeled unmyelinated axons is demonstrated. Note the electron dense immunoprecipitate coating microtubules (small arrow). Scale bar - 0.15 ~tm. B A TH-positive varicosity containing large dense core vesicles (arrow) is shown. Note unmyelinated axons (clear arrows) on either side of the labeled profile. Scale b a r - 0.19 gin. C Typical synaptic relationship between a TH-positive axon terminal and small dendrite is demonstrated. Note the widened synaptic cleft and symmetrical postsynaptic specialization (large arrow). Small, round and clear synaptic vesicles are discernable (small arrow). Scale bar - 0.24 ~tm, D A TH-positive axon terminal con-

taining round (large black arrow) and elliptical-shaped (small black arrow) vesicles. Symmetric postsynaptic specializations are present (clear arrowheads). Scale bar - 0.22 gin. E A TH-positive axodendritic terminal containing varying sizes of round, agranular synaptic vesicles (small and large black arrows). The precise morphology of the synaptic specialization is not obvious. Note the adjacent nonlabeled axon terminal containing a large DCV and numerous round and oval vesicles. Scale b a r - 0.25 gm. F A TH-positive axodendritic terminal containing a large DCV with a dense central core and outer unstained rim (arrow). Small, round vesicles and a symmetric synaptic specialization are evident (clear arrow). Scale bar - 0.3 gm

172 bers o f large (55-90 gm) dense core vesicles ( D C V - Figs. 3B). Neither u n m y e l i n a t e d axons n o r varicosities were associated with a synaptic specialization or m a d e synaptic c o n t a c t with a n y p o s t s y n a p t i c structure (Figs. 2A; 3A, B). I m m u n o p r e c i p i t a t e was seen t h r o u g h o u t the axoplasm a n d was associated with D C V and microtubules (Figs. 3A, B). N o i m m u n o r e a c t i v e myelinated axons were observed. I m m u n o r e a c t i v e a x o n terminals were seen in all regions o f X I I but were e n c o u n t e r e d less frequently t h a n labeled preterminal axons and varicosities. TH-positive terminals a c c o u n t e d for only 43.5% o f the total n u m b e r o f labeled profiles identified in this study. The m a j o r i t y

o f labeled a x o n terminals (71.8 %) synapsed on dendrites, while the remaining terminals synapsed on s o m a t a (25.4%) a n d on non-labeled a x o n terminals (2.7%). T H positive terminals synapsed mainly on small ( < 2 gin) and m e d i u m (2-3.5 gin) sized dendrites; less frequently on large, p r o x i m a l ( > 3.5 gin) dendrites. The m o r p h o l o g y o f labeled a x o n terminals was variable. Terminals ending o n small-to-medium sized dendrites typically contained n u m e r o u s densely p a c k e d small, r o u n d and clear vesicles (20-50 nm) and were associated with either a symmetrical or no postsynaptic specialization (Figs. 2B; 3C, E ; 4 A - D ) . H o w e v e r , occasionally an axodendritic terminal associated with a small

Fig. 4. A Two TH-positive axon terminals (black arrows) in contact with a single hypoglossal dendrite are indicated. Non-labeled terminals synapsing on the same dendrite are indicated (clear arrows). Scale bar - 0.67 lam. B A single TH-positive axon terminal in apparent contact with different postsynaptic structures (D1, D2) is demonstrated. The synaptic cleft of one terminal is clearly defined (large arrow), while the remaining cleft is not obvious (small arrows). Scale bar - 0.7 lam. C A typical TH-positive axon terminal (arrow) synapsing on a proximal dendrite (Pr). X 15,800. Inset -

higher magnification of the same labeled axon terminal. Note the symmetrical postsynaptic specialization (arrowhead), mitochondria (m) and small, round agranular vesicles. Scale bar - 1.15 jim; 0.26 gm (inset). D A TH-positive profile in contact with a proximal dendrite (Pr) that appears to be morphologically more related to a preterminal axon (compare with Figs. 3A) than to a typical axon terminal (compare with Figs. 3E, F). There is no discernible synaptic specialization (arrow). Scale bar - 0.17 ~tm

173

asymmetrical postsynaptic density was observed (not shown). Axodendritic terminals occasionally contained large (55-90 nm) DCV (Fig. 3F) and/or a few ellipticshaped vesicles (Fig. 3D). In lightly stained terminals DCV appeared to have a small dense core (40-45 rim) surrounded by an outer clear rim (Figs. 3F), while others appeared more completely stained (Fig. 4D). Not all terminals containing DCV were immunopositive (Fig. 3E) and many terminals contained varying sizes of small, round agranular vesicles (Fig. 3E). A single unlabeled dendrite was often found engaged synaptically by multiple TH-positive terminals (Fig. 4A), and conversely, a single labeled termi-

nal was frequently observed in close contact with two adjacent unlabeled dendrites (Fig. 4B). Axodendritic terminals ranged in size from 0.31-1.36x0.19-0.88 gm (J~ = 0.77 x 0.40 ~tm). A few labeled axon terminals (N= 18) were found synapsing on proximal dendrites. These terminals exhibited the same morphology as those terminating on smaller dendrites, i.e., mainly small, round, agranular vesicles accompanied by a symmetrical or no synaptic specialization (Fig. 4C). However, in some cases labeled profiles characteristic of preterminal axons were seen adjacent to proximal dendrites (Fig. 4D), but a synaptic

Fig. 5. A The soma of a protrusor motoneuron in the caudoventromedial q u a d r a n t of XII is contacted by several TH-positive profiles (arrows). N u - nucleus. Scale bar - 0.90 gin. B A TH-positive axosomatic terminal exhibiting a presynaptic thickening (large arrow) and small, round vesicles (small arrow). Scale bar 0.17 gm

174 cleft was not apparent and no synaptic specialization could be discerned (Fig. 4D). T H - p o s i t i v e axon terminals were f o u n d synapsing on s o m a t a o f m o t o n e u r o n s (Fig. 5) in all regions o f X I I . A x o s o m a t i c terminals ranged in size f r o m 0 . 3 5 - 1 . 1 4 x 0 . 1 - 0 . 5 3 g m (X = 0.69 x 0.29). These terminals typically contained round, agranular and a few elliptical-shaped vesicles (Fig. 5). These terminals were often associated with a presynaptic m e m b r a n e thickening (Fig. 5B) or, in some cases, with a symmetrical postsynaptic specialization. Multiple contacts were often seen

on large m o t o n e u r o n s (Fig. 5A), particularly in the caudoventromedial quadrant. Axon terminals positive for T H were occasionally found synapsing on nonlabeled axon terminals (Fig. 6). Labeled terminals (0.75 x 0.37 gm) contained numerous small, round, agranular vesicles and were associated with asymmetric postsynaptic specializations (Fig. 6B). The m o r p h o l o g y of recipient postsynaptic terminals was similar to that of the TH-positive presynaptic terminal. In each case the postsynaptic terminal was in contact with an unlabeled dendrite. In one instance, the unlabeled

Fig. 6. A Electron micrograph showing two TH-positive axon terminals in relation to a single dendrite (D1). One terminal (1) synapses directly on D 1 (axodendritic), while the other labeled terminal (2) synapses on an unlabeled terminal (*) forming an axoaxonic synapse (black arrow). The unlabeled terminal is, in turn, presynaptic to D1 (clear arrow). D2 dendrite. Scale bar - 0.42 gm. B Higher magnification of the axoaxonic synapse in A. Note the multiple asymmetric postsynaptic specializations (large arrows) and numerous small, round, agranular synaptic vesicles (small arrow), m = mitochondria. Scale bar - 0.22 gm

175 dendrite was contacted by another TH-positive terminal (Fig. 61). Controls Light microscopic analysis of flat-embedded sections that were incubated in carrier without primary antibody or in carrier saturated with rabbit serum resulted in no immunostaining in XII and throughout the medulla (Fig. 1C). Ultrathin sections cut through the first few microns of control tissues and examined in the EM showed no electron-dense immunoprecipitate. Discussion

The present study utilized correlative light and electron microscopy in conjunction with immunocytochemistry to identify the ultrastructure of CA-containing axon terminals in XII of the rat. The major findings of this study were the following: 1) Most CA profiles were nonsynaptic unmyelinated axons and varicosities (Table 1); 2) Labeled axon terminals synapsed on somata, dendrites and non-labeled axon terminals, although primarily on dendrites (Table 2); 3) The morphology of labeled terminals was variable; and 4) Labeled dendritic processes were identified in XII. These results confirm and extend previous reports regarding the CA innervation of XII (Swanson and Hartman 1975; Levitt and Moore 1979; Palkovits and Jacobowitz 1979; Kalia et al. 1985; Aldes et al. 1988a, c). The validity of these observations is based upon the utilization of a well characterized, affinity purified, antibody to TH (Haycock 1987) and the negative findings from control experiments (Fig. 1C). The morphology and distribution patterns of TH immunoreactivity observed in flat-embedded sections are consistent with our previous light microscopic results (Aldes et al. 1988a, c). The present study is the first to provide an ultrastructural analysis of CA-containing axon terminals in XII. Since we used TH as a probe for CA, and TH is the rate limiting enzyme in the biosynthesis of all CA (Nagatsu et al. 1964; Levitt et al. 1965), the precise nature of the CA identified in this study is uncertain. It is probable, however, that most, if not all, of the labeled profiles identified herein are NE since it is well established that NE is the principal CA in XII (Levitt and Moore 1979; Aldes et al. 1988c). This is consistent with findings in other motoneuron cell groups (Zivin et al. 1975; Skagerberg et al. 1982). Relationships to other motoneuron groups Previous investigators have used antisera to dopaminebeta-hydroxylase (DBH l k e y s o n et al. 1983; Kojima et al. 1985) or the false neurotransmitters 5- and 6-hydroxydopamine (5-OHDA, 6-OHDA - Card et al. 1986; 5-OHDA - Mizukawa and Takeuchi 1982) to study NE terminals in other lower motoneuron groups. Our results

are in general agreement with findings from these studies, although some differences were found. For example, Kojima et al. (1985) reported that DBH-positive terminals in rat lumbosacral ventral horn contained small, round and occasionally large DCV, terminated on dendrites and somata but mainly on dendrites (67.9 % versus 15.1%), and occasionally exhibited synaptic specializations, some of which were symmetrical. We observed 71.8 % and 25.4 % of synaptic profiles ending on dendrites and somata, respectively, and that most were associated with a symmetric or no synaptic specialization. Akeyson et al. (1983) reported in abstract form that DBH-positive terminals also ended predominantly on dendrites in rat ventral horn. However, in contrast to our findings, they found that axodendritic terminals formed mainly asymmetric membrane specializations. In the present study we observed only a few asymmetric specializations associated with axodendritic terminals, whereas all labeled axoaxonic terminals were characterized by this morphology (Fig. 6). This discrepancy is likely due to differences in methodology and/or sampling, although differences in functional organization between XII and the ventral spinal cord cannot, at this time, be excluded. Using false neurotransmitters, Card et al. (1986) in rat motor trigeminal nucleus and Mizukawa and Takeuchi (1982) in the cat ventral horn also found NE terminals containing mainly small, round vesicles ending predominately on dendrites. Card et al. (1986) reported that almost half of labeled terminals were associated with a synaptic specialization, but did not specify the type. Mizukawa and Takeuchi (1982), however, reported that the majority of axodendritic terminals were associated with an asymmetric postsynaptic density. In this context, it is relevant to note that serotonin, the other major monoamine in XII (Aldes et al. 1988a), was recently shown to terminate predominately on dendrites and to form pronounced asymmetric synaptic specializations (Aldes et al. 1989). Since Mizukawa and Takeuchi (1982) used 5-OHDA as their sole probe, and 5-OHDA could label both NE and 5-HT (Richards and Tranzer 1970; Mizukawa and Takeuchi 1982), one must conclude that their results include both monoamines and cannot, therefore, be compared with ours with respect to synaptic specialization. Synaptic morphology The morphology of labeled axon terminals was variable. Axodendritic terminals contained mainly round, clear synaptic vesicles and formed either symmetrical or no postsynaptic specializations (Figs. 2B; 3C-F; 4). A few were associated with a small asymmetric postsynaptic density (not shown), while others contained ellipticshaped vesicles (Fig. 3D). Terminals ending on somata also contained densely packed small, round vesicles and a few elliptical-shaped vesicles (Fig. 5B), but in contrast to those ending on dendrites, were often associated with a presynaptic membrane thickening (Figs. 5B). By contrast, axoaxonic terminals were associated with multiple asymmetric postsynaptic densities and contained only

176 small, round, agranular vesicles. The variability observed among synaptic terminals may be attributed to several factors. First, the immunoprecipitate often obscured the fine details of synaptic morphology. This is a well recognized limitation of the immunoelectron technique (O1schowska et al. t981; Pasik et al. 1982; Kojima et al. 1985). Second, due to the necessary constraints of the immunocytochemical method, ultrastructural detail was often not optimally preserved. Third, differences in synaptic morphology may be a reflection of multiple NE inputs. The possibility is plausible since preliminary results suggest that NE-XII afferents are derived from several sources including the subceruleal area, paralemniscus and the nucleus solitarius (Aldes and Chapman, in preparation). And, fourth, as noted previously, since we used antibody to TH, we cannot exclude the possibility that some labeled terminals are DA and/or E. However, this possibility seems remote since the vast majority of CA innervation of XII is NE (Levitt and Moore 1979; Aldes et al. 1988c). Given the sparseness of DA and/or E in XII, the likelihood of consistently finding such labeled profiles is difficult to conceive.

that traverse XII (Odutola 1976; Scheibel and Scheibel 1970; Kalia et al. 1985; Aldes et al. 1988a, c). In this context it is of interest that we have identified CApositive dendrites in XII (Fig. 2A). Although the precise origin of these processes remains speculative, one can see at the light microscopic level presumptive dendritic processes of CA-positive neurons emanating from neurons in the dorsal vagal and solitarius nuclei extending into XII (Aldes et al. 1988a, c; Kalia et al. 1985). This suggests that specific input signals directed at XII motoneurons may be accessed by dorsal vagal and/or solitarius neurons. This type of relationship must be considered in future studies that attempt to understand the functional organization of these brainstem nuclei. Axoaxonic synapses were observed in XII (Fig. 6). To our knowledge this is the first evidence of such formations in rat XII. That CA-positive axon terminals synapse on non-labeled axons suggest a mechanism for presynaptic regulation of incoming non-CA signals. Of further interest is the finding of CA-positive terminals contacting the same dendrite which is the target of CAregulated presynaptic control (Fig. 6A). Further investigation of these relationships is warranted.

Synaptic distribution Synaptic versus nonsynaptic Previous results have demonstrated that CA immunoreactivity is greatest among protrusor motoneurons in the caudoventromedial quadrant of XII (Aldes et al. 1988a, c), and the present flat-embedded material supports our earlier observations (Fig. la). We have now confirmed that this motoneuron subgroup is synaptically engaged by CA-containing axon terminals (Fig. 5), and have further demonstrated that individual motoneurons in this group typically receive multiple CA inputs (Fig. 5A). Furthermore, it is noteworthy that some motoneuron somata in the dorsal (retrusor) district of XII also received TH-positive synaptic contacts. This finding was not surprising since light microscopic observations clearly show that all regions of XII contain CA-positive profiles (Aldes et al. 1988a, c; Fig. 1A). Our results also show that CA-containing axon terminals end mainly on dendrites (Table 2). This finding is in complete accord with those previously reported for CA in other motor nuclei (Akeyson 1983; Kojima et al. 1985; Card et al. 1986; Mizukawa et al. 1982) and for serotonin in XII (Aldes et al. 1989). Yet, we cannot state with certainty that labeled terminals end preferentially on dendrites of protrusor (ventral) or retrusor (dorsal) motoneurons since there is extensive intermingling of these processes throughout XII (Cajal 1909; Odutola 1976; Wan et al. 1982). Thus, dendrites located ventrally and dorsally may be derived from either protrusor or retrusor motoneurons. This issue is of obvious importance to understanding the relationships between functionally antagonistic protrusor/retrusor motoneurons and is currently being investigated. We cannot either exclude the possibility that CAcontaining axon terminals contact dendrites of intrinsic interneurons (Boone and Aldes 1984; Takasu and Hashimoto 1988), or dendritic processes of adjacent neurons

We found that 43.5% of immunoreactive TH profiles formed definitive synaptic contacts, while 55.2% were nonsynaptic unmyelinated axons and varicosities (Table 1). Although all regions of XII were investigated and over 500 labeled profiles were studied in detail, our estimates must be considered conservative due to sampling problems associated with ultrastructural investigations; a problem compounded by immunocytochemistry vis-avis limited antibody penetration and the deliterious effects of detergents on membrane ultrastructure used to facilitate antibody penetration. Nevertheless, the proportion of synaptic versus nonsynaptic labeled profiles found in this study is within the range reported by others using different techniques. Specifically, the number of labeled synaptic contacts in this study was less than that reported by Akeyson et al. (1983; > 50%) and Mizukawa and Takeuchi (1982; 60%) in the ventral horn or rat and cat, respectively, and by Card et al. (1986) in rat motor trigeminal nucleus (50%), but is greater than that reported by Kojima et al. (1985) in the rat lumbosacral ventral horn (20 %). Although this discrepancy may be attributed to methodological, sampling and/or data analysis differences, it is particularly relevant to note that numerous other studies have demonstrated variable degrees of NE synaptic specificity among different brain regions (Pickel et al. 1976; Descarries et al. 1977; Beaudet and Descarries 1978; Cimarusti et al. 1979; Olschowka et al. 1981) ranging from greater than 58% in the diencephalon, cerebellum and limbic cortex (Olschowka et al., 1981) to less than 5% in the neocortex (Descarries et al. 1977). Collectively, these results suggest a unique functional organization with regard to NE control for different neural sites. This concept is further buttressed by the recent findings of Grzanna et al. (1987) who showed that

177 the N E innervation of the facial and trigeminal m o t o r nuclei is derived from different subsets of N E neurons in the A7 and A5 CA cell groups. It is therefore quite conceivable that X I I also receives innervation from subgroups of N E neurons that are largely, if not exclusively, distinct from those innervating other cranial nerve m o t o r nuclei, and that this difference may be reflected in the proportion of synaptic versus non-synaptic labeled profiles. As noted previously, preliminary results from this laboratory indicate that N E - X I I afferents are derived from multiple sources (Aldes and Chapman, in preparation), and studies are currently underway to determine the relative contribution of N E axon terminals to X I I from each of these sites.

Functional considerations The precise effects of CA on X I I motoneurons are unknown. In slice preparation, N E was found to exert an excitatory effect on X I I m o t o n e u r o n activity (Gregg and Carpenter 1982). N E has been reported to have excitatory, inhibitory and complex effects on spinal motoneuron activity (Strahlendorf et al. 1980; White and N e u m a n 1980, 1983; Fung and Barnes 1981; Chan et al. 1986; Wohlberg et al. 1986). The presence of morphologically distinct CA-positive terminals in X I I and their ending on dendrites, somata and axon terminals provides the morphological substrates that subserve these effects. Furthermore, the presence of both nonsynaptic and synaptic immunoreactive profiles in X I I suggests a possible dual functional role for CA in controlling XII m o t o n e u r o n activity. C A released from preterminal axons and/or varicosities may modulate X I I motoneurons nonspecifically, or C A may serve as a definitive neurotransmitter derived from axon terminals whose effects are directed onto specific postsynaptic structures in X I I via traditional synaptic engagement (Pickel et al. 1976; Descarries et al. 1977; Beaudet and Descarries 1978). These two mechanisms, however, are not mutually exclusive. Finally, the preponderance of labeled profiles, both synaptic and nonsynaptic, a m o n g dendritic fields throughout X I I is noteworthy. Dendritic bundles have been identified in X I I (Cajal 1909; Lorente de N o 1947) and, as in the ventral spinal cord, presumably provide a substrate for integrating divergent synaptic influences (Scheibel and Scheibel 1970). Interestingly, dendritic bundles of phrenic motoneurons are recipients of CA innervation (Bellinger et al. 1984) and may serve an important role in the synchronization of respiration. A similar argument could be made for X I I since the base of the tongue is intimately related to the inspiratory phase of respiration (Sauerland and Mitchell 1970, 1975; Brouillette and Thach 1979, 1980).

Acknowledgement. This research was supported by NIH grant NS-22686 (L.D. Aldes).

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Note added in proof

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