An Electron Microscope Study of Motoneurones and lnterneurones in the Cat Abducens Nucleus Identified by Retrograde lntraaxonal Transport of Horseradish Peroxidase 12 ROBERT F. SPENCER AND PETER STERLING Department ofAnatomy and Institute o f Neurological Sciences, Uniuersrty of Pennsyluania, The School of MedicinelG3, Philadelphia, Pennsyluania 19104

ABSTRACT Abducens motoneurones innervating the lateral rectus muscle and abducens interneurones projecting to the oculomotor nucleus have been studied in the cat by light and electron microscopy following labelling with horseradish peroxidase. Motoneurones ranged from 15-60 p m in diameter and typically contained circular nuclei with smooth or slightly irregular nuclear membranes and extensive cisternae of granular endoplasmic reticulum. Interneurones ranged from 25-50 p m in diameter and comprised two cytologically distinct cell types. One type was fusiform in profile and contained nuclei with fluted or deeply invaginated nuclear membranes and poorly developed granular endoplasmic reticulum. The other interneurone type resembled the motoneurones cytologically, having multipolar or circular profiles, smooth or slightly irregular nuclear membranes, and well developed cisternae of granular endoplasmic reticulum. The densities with which boutons were distributed on the somata of motoneurones and interneurones were similar, as were the ratios of boutons containing spheroidal or flattened synaptic vesicles. Motoneurones varied significantly in the density with which boutons were distributed on their somata, and had greater densities on their dendrites than on their somata. Density was, furthermore, not correlated with motoneurone soma diameter. The proportion of spheroidal or flattened vesicle boutons on the somata of motoneurones was almost equal, with comparatively little variation between individual motoneurones. Motoneurone dendrites, however, with the exception of lateral dendrites, had a relatively higher proportion of boutons containing spheroidal synaptic vesicles. The lateral dendrites, which extend toward the medial vestibular nucleus, a known source of inhibitory afferents to the abducens motoneurones, had a higher proportion of boutons containing flattened vesicles. The results indicate t h a t abducens motoneurones and interneurones cannot be distinguished by any simple morphological criteria. The somadendritic distribution of boutons on motoneurones appears to be governed by certain broad rules. The cat abducens Offers particular advantages for anatomical inVeStigatiOnS O f mammalian motoneurones. I t contains neurones that il"V.?rvate primarily a single muscle, the lateral rectus, rather than a complex admixture Of as in the cord (e.g., Romanes, '51; Sterling and Kuymotoneuronesj

J. COMP. NEUR., 176: 65-86.

'

In honour of his sixtieth birthday, we dedicate this paper to Pro. fessor James M. Sprague. who began his neuroanatomical career with experimental studies of motoneurones and interneurones. This study was supported by U.S.P.H.S. Postdoctoral Research Fellowship EY00361,Research Grant EY00828, and Vision Research Center Grant EY05012.from the National Eye Institute. Address for reprint requests. Department of Anatomy, P 0. Box 906, Medical College of Virginia, Virginia Commonwealth Unwersity, MCV Station, Richmond, Virginia 23298.

65

66

ROBERT F. SPENCER AND PETER STERLING

pers, '67) and other cranial nerve motor nuclei. Several important sources of direct afferents to the abducens nucleus have been identified: the contralateral and ipsilateral vestibular nuclei (Lorente de No, '33; Szentagothai, '50; Schaeffer, '65; McMasters e t al., '66; Precht et al., '67, '69; Richter and Precht, '68; Baker et al., '69; Tarlov, '70; Gacek, '71; Maeda et al., '71, '72; Highstein, '731, the contralateral oculomotor nucleus (Maciewicz et al., '761, and the pontine reticular formation (Graybiel, '75; Buttner-Ennever and Henn, '76; Highstein et al., '76). These sources are well enough separated from the abducens nucleus that their pathways can be labelled by degeneration and autoradiographic methods. This motor nucleus, therefore, presents a n excellent site at which to study the detailed distribution of terminals derived from specific afferent sources that converge upon a final common path. The abducens nucleus is not, however, perfectly homogeneous. In the first place, its neurones exhibit differences in both size and shape (van Gehuchten, 1898; Ramon y Cajal, '09; Fuse, '12; Taber, '61). Second, it contains, in addition to motoneurones, a population of interneurones that project to the medial rectus subdivision of the contralateral oculomotor complex (Graybiel and Hartwieg, '74; Baker and Highstein, '75). Third, the motoneurones are themselves somewhat heterogeneous. "Tonic" and "phasic" motoneurones have been distinguished by differences in their resting discharge rates (Precht et al., '69; Henn and Cohen, '721, axonal conduction velocities (Yamanaka and Bach y Rita, '68; Precht et al., '691, and behavior during eye movements (Schaeffer, '65; Yamanaka and Bach y Rita, '68; Henn and Cohen, '72; see, however, Schiller, '70; Robinson, '70; Keller and Robinson, '72). These differences probably correspond, a t least in part, to the known anatomical (Peachey, '70; Alvarado and Horn, '75) and physiological (Bach y Rita, '71) differences between muscle fibers in the extraocular muscles (cf., Lennerstrand, '75; Goldberg et al., '76). Clearly, before the organization of extrinsic afferents can be studied in detail, a way must be found to recognize a t the ultrastructural level the various types of cells within the nucleus. In the present study, the lateral rectus motoneurones and interneurones within the abducens nucleus have been separately labelled using the technique of retrograde

intraaxonal of horseradish peroxidase (Kristennson and Olsson, '71; La Vail and La Vail, '72; Gacek, '74; Graybiel and Hartwieg, '74) and examined by light and electron microscopy. We hoped to identify differences between the neurones in cytology and synaptology that would be useful in future studies. The results indicate that motoneurones and interneurones cannot be distinguished on the basis of their normal cytology or synaptology alone. We did find, however, significant differences within the motoneurone population in the density and distribution of synaptic endings over the soma-dendritic surface. MATERIALS AND METHODS

Normal animals Four adult cats were prepared for the study of the normal cytology and synaptology of abducens neurones. Each cat was anaesthetized with Nembutal. As the thorax was opened, artificial respiration was initiated with a mixture of 95% oxygen and 5%carbon dioxide. Heparin sodium (2,000 I.U.) and sodium nitrite (10 mg) were injected intravenously via a catheter in a femoral vein. The cat was then perfused through the ascending aorta with 1,500 ml of fixative solution containing 1%paraformaldehye and 1.25% glutaraldehyde in 0.12 M phosphate buffer with 0.002% CaC1, a t pH 7.4, over a 30-minute period. The head was removed two hours later and immersed in cold fixative for 12 to 18 hours. The brainstem was blocked stereotaxically, and 100-pm coronal sections through the abducens nucleus were cut with a Vibratome and collected serially in a phosphate rinse buffer. The sections were postfixed for one hour with 2% osmium tetroxide in phosphate buffer and stained en bloc with 0.5% or 2.0%uranyl acetate (Karnovsky, '67). The material was dehydrated in methanol and propylene oxide, and embedded in Epon 812. Semithin (1 pm) sections were obtained with a n ultramicrotome using glass knives and stained with toluidine blue. Ultrathin (60-80 nm) sections were obtained following every fifth semithin section with a diamond knife, mounted on formvar-coated single-slot grids, stained with uranyl acetate and lead citrate (Reynolds, '631, and examined and photographed with a JEOL JEM-12OB electron microscope. Experimental animals Four adult cats were used for the iden-

ABDUCENS MOTONEURONES AND INTERNEURONES

tification of motoneurones innervating the right lateral rectus muscle. In each cat anaesthetized with Nembutal, the muscle was exposed by dissection. Three to five injections of approximately 20 p1 of 5% HRP (Type VI, Sigma Chemical Co.) in Ringer's solution were made with a Hamilton microsyringe along the rostro-caudal extent of the muscle. In one cat, the lateral slip of the retractor bulbi muscle was isolated from the overlying lateral rectus muscle and was similarly injected with HRP, since physiological studies have suggested that motoneurones innervating this muscle in the cat are also located in the abducens nucleus (Bach y Rita and Ito, '65). Four adult cats were used for the identification of the interneurones in the abducens nucleus that project to the oculomotor complex. In each cat, four injections of 0.050.15 p1 each of 25% HRP in 0.9% saline were made stereotaxically a t 0.75-mm intervals along the rostro-caudal extent of the left oculomotor nucleus over a 90-minute period. The injections were made with a Hamilton microsyringe following the procedure described in detail by Edwards ('72). The cats were sacrificed following a survival period of 24 to 48 hours by intracardial perfusion of the double aldehyde fixative solution a s previously described. Serial 50-pm coronal sections through the abducens, trochlear, and oculomotor nuclei were obtained with the Vibratome and processed for the histochemical demonstration of HRP (Graham and Karnovsky, '66; La Vail and La Vail, '74). Sections were washed in cold phosphate rinse buffer and then incubated in a medium consisting of 0.05%3,3'diaminobenzidine tetrahydrochloride (DAB) and 0.01%hydrogen peroxide (H,O,) in phosphate buffer, pH 7.4, for one hour at 4°C with intermittent agitation. Following incubation, the sections were washed in cold rinse buffer and processed for electron microscopy as described above. The injection sites into the oculomotor complex were verified from 40-pm coronal sections cut with a freezing microtome and incubated in the DAB-H,O,-phosphate buffer medium. Sections were mounted on albumencoated slides, and alternate sections were counterstained with thionine.

67

nifications in stained ultrathin sections because the differences between HRP vesicles and the normal complement of lysosomes and lipofuscin granules are subtle. Our procedure, therefore, was first to identify labelled cells in semithin sections with the light microscope (fig. 8). The same cells were then photographed in the adjacent ultrathin sections with the electron microscope a t a magnification of x 1,400 (fig. 10).Each of these neurones could be located on a low magnification ( x 90) electron micrograph of the ultrathin sections (fig. 9) and matched to the same neurones in the light micrograph of the adjacent semithin section. RESULTS

Distribution and morphology of the motoneurones and interneurones Following injections of the right lateral rectus muscle, many densely-labelled neurones in the right abducens nucleus contained the characteristic brown HRP granules. These formed a ring around the cell nucleus and frequently filled the cytoplasm of the soma and proximal dendrites (fig. 8). We compared the numbers of labelled and unlabelled neurones in semithin sections, each separated by approximately 50 pm, and, in the most successful experiment, estimated that approximately 85%of the cells in the ipsilateral abducens nucleus were labelled. These cells were distributed throughout the rostro-caudal extent of the nucleus and tended to be arranged in clusters in the central part of the nucleus and loosely toward the periphery. There was no segregation of motoneurones by size within the nucleus; that is, large motoneurones were not preferentially located dorsally and small motoneurones ventrally, or vice-versa. There were no labelled cells along the rootlets of the abducens nerve over its ventral course through the reticular formation. No labelled cells were found in either the opposite abducens nucleus, nor in the trochlear or oculomotor nuclei of either side. This suggests that the HRP injections were effectively confined to the lateral rectus muscle. The motoneurone somata ranged from approximately 15-60 p m in mean diameter, with an apparently unimodal size distribution (fig. 1).The largest cells (40-60 pm) had multipoElectron microscope identification of HRPlar or triangular perikarya; the medium (25labelled neurones 40 pm) and small (15-25 p m ) cells were It is difficult to identify HRP-labelled neu- typically circular or pyriform in profile. The rones in the electron microscope a t low mag- nucleus of the cell was in all cases circular in

68

ROBERT F. SPENCER AND PETER STERLING

INTERNEURONES

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SOMA DIAMETER (urn) Fig. 1 Distribution of mean diameters (average of longest and shortest diameters measured through nu cleolar plane) of 121 motoneurones and 34 interneurones.

profile and its membrane smooth in contour. The neurones were filled with the usual cytoplasmic organelles (mitochondria, Golgi apparatus, neurofilaments and microtubules, polyribosomes, lysosomes, multivesicular bodies, and occasional lipofuscin granules), and had, as motoneurones do, elaborate cisternal arrays of granular endoplasmic reticulum (fig. 10). In the experiment in which the lateral slip of the retractor bulbi muscle was injected with HRP, eleven labelled motoneurones, 3452 p m in diameter, were found scattered through the mid-portion of the ipsilateral abducens nucleus, and, a t this level, accounted for approximately 30% of the cells. The interneurones that project to the oculomotor complex showed the same intracellular pattern of granular HRP label as the motoneurones (fig. 11).By comparing the numbers of labelled and unlabelled cells in semithin sections, each separated by approximately 50 p m , we estimated that approximately 25% of the cells in the abducens nucleus were labelled in the most successful experiment. The interneurones, like the motoneurones, were distributed uniformly throughout the rostrocaudal extent of the nucleus. No interneurones were found scattered in the immediately surrounding reticular formation, not in the rostra1 portion of nucleus prepositus hypo-

glossi. Some cells were found in the abducens nucleus ipsilateral to the side of the injection. This in no way contradicts the finding of Graybiel and Hartwieg ('74) that the projection is exclusively contralateral. In order to get as complete labelling of the contralateral abducens nucleus as possible, our injections into the oculomotor complex were deliberately large and were, therefore, to some extent bilateral. The size range of the interneurone somata was approximately 25-50 p m , somewhat smaller than the range for the motoneurones (fig. 1).Some of the interneurones were consistently distinguishable cytologically from the motoneurones. These interneurones had fusiform perikarya, nuclear envelopes that were consistently either fluted or deeply invaginated, and granular endoplasmic reticulum that was poorly developed (fig. 12). Other interneurones resembled the motoneurones cytologically, having multipolar or circular profiles, smooth or only slightly irregular nuclear membranes, and well developed cisternae of granular endoplasmic reticulum (fig. 13). Possibly there are two different classes of interneurones in the cat abducens nucleus. Synapses on motoneurones and interneurones

The cell bodies and dendrites of both

ABDUCENS MOTONEURONES AND INTERNEURONES

motoneurones and interneurones were abundantly covered with boutons that form synaptic contacts typical of chemical synapses. A distinction between boutons containing predominantly spheroidal vesicles and those containing predominantly "flattened" (pleiomorphic or ellipsoidal) synaptic vesicles could be made both in normal material and in material incubated for the HRP reaction product (fig. 14). Marked asymmetries in membrane densification at the sites of synaptic contact, such as found in the cerebral cortex in association with boutons containing spheroidal synaptic vesicles (Colonnier, '681, were not consistently apparent. Although we suspect that the boutons containing flattened vesicles are a heterogeneous group, we made no attempt a t subclassification, feeling that the distinctions which might be made are in this material subtle and vulnerable to minor variations in preparative technique (Walberg, '66; Bodian, '71; Valdivia, '71; Paula-Barbosa, '75). Similarly, although we observed differences in size, content of mitochondria, and arrangements of neurofilaments in both spheroidal and flattened vesicle boutons, these seemed too dependent on the plane of section through the bouton to be reliable guides for classification. For example, in figure 15 a single bouton containing flattened synaptic vesicles can be seen making multiple synaptic contacts on a motoneurone soma. Although vesicle shape is the same a t all sites of synaptic contact, the axoplasmic organelles differ both qualitatively and quantitatively a t any particular point along the synaptic ending. Furthermore, the two smaller boutons on either side of the large bouton also contain flattened synaptic vesicles, and, therefore, are possibly extensions of the large bouton, but may only appear discontinuous because of the plane of section. Additional distinctions between axosomatic and axodendritic boutons, therefore, await the completion of experimental labelling of the terminals derived from specific afferent sources. We had anticipated finding boutons with gap junction contacts because of a report that cat abducens motoneurones are electronically coupled (Gogan et al., '75). None were found, however, despite a n extensive search both in normal material and material incubated for the HRP reaction product. If boutons with gap junction contacts exist within the cat

69

abducens nucleus, they must be smaller than in other species with electrically coupled oculomotor neurones (Waxman and Pappas, '70; Sterling, '77). Possibly, they could be on the motoneurone axon hillocks or initial segments, which we encountered only rarely. Axo-axonic synapses were not found in the cat abducens nucleus. This is in striking contrast to the cat spinal motor nuclei, where axo-axonic contacts are abundant (Conradi, '69; Saito, '72). I t is, however, consistent with the fact that they are absent in the abducens nucleus of the teleost (Sterling, '77). The present negative finding seems to support an earlier suggestion (Sterling, '77) that axo-axonic synapses in motor nuclei are associated with recurrent or autogenic reflexes, which are absent or weak in the oculomotor system (McCouch and Adler, '33; Fuchs and Luschei, '71; Keller and Robinson, '71). There is a report, however, of axo-axonic synapses in the oculomotor complex of the cat and monkey (Waxman and Papas, '70). Dendritic profiles were frequently encountered in direct apposition to neuronal perikarya. In some instances they resembled mere labile (Brightman and Reese, '69) or "casual" (Sotelo et al., '74) appositions. In others, however, a desmosome-like plaque was apparent, with flocculent, electron-dense material aggregated along the pre- and post-junctional membranes (fig. 16).

Distribution of boutons on the neuronal surfaces We compared the density with which boutons are distributed over the somatic surfaces of 1 2 1 motoneurones and 34 interneurones sectioned through the nucleolar plane. The number of synapses/100 p m Zof somatic membrane was calculated as the square of the number of synapsesIl0 linear p m on the electron micrographs. Densities calculated in this way can thus be compared to direct measurements of synaptic density on spinal motoneurones made with the light microscope (e.g., Gelfan and Rapisarda, '64). The average density calculated from single sections was remarkably similar for both motoneurones (4.2 f 3.4 synapses/100 pm2) and interneurones (4.6 f 3.8 synapses/100 pm2).The range of densities was also similar for these two cell types (0.2-17.6 synapses/100 pm2 for motoneurones; 0.1-10.8 synapses/100 p m 2 for interneurones). As shown in figure 2,

70

ROBERT F. SPENCER AND PETER STERLING 18* 1

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NEURONE DIAMETER Fig. 2 Distribution of densities of axosomatic boutons determined from single sections through nucleolar plane of 121 motoneurones (MN) and 34 interneurones (IN). Note t h a t distributions for motoneurones and interneurones overlap almost completely.

the distribution of synaptic densities of motoneurones and interneurones overlaps completely. No differences were noted between the fusiform and circular or multipolar interneurones. In order to determine whether the density differences between individual motoneurones were real or merely sampling errors, we studied 18 labelled motoneurones in more detail. Each cell was photographed in three to seven sections, each section separated from the next by approximately 5 pm. The variation in average density between cells was considerable, 1.2-10.1 synapses/100 pm2. As seen in table 1, the variation between sections through the same cells was, in some cases, substantial. Nevertheless, it is clear from figure 3 that there are many cells for which the standard errors of the mean do not overlap; that is, there are real differences between motoneurones in total synaptic density. Den-

sity was, furthermore, not correlated with motoneurone soma diameter. We also calculated bouton densities for 36 primary dendrites that extended 40-110 p m from the cell bodies of 16 motoneurones studied in detail. The mean densities were substantially greater for the dendrites (6.9 2 2 . 7 synapses/100 pm2) than for the cell somata. There was no association between the densities on dendrites and the densities on the somata from which the dendrites were derived; that is, somata with low bouton densities could have dendrites with either high or low densities, and vice-versa. Bouton densities of different dendrites from the same motoneurone were sometimes similar and sometimes quite different. As shown in figure 4, there was no obvious pattern to the differences in synaptic density between dendrites of different orientations.

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SOMA SYNAPSES/IOO urn2 Fig. 4 Plot of bouton densities on motoneurone dendrites vs. bouton densities on motoneurone somata. All points below and to right of dotted line represent dendritic densities less than respective somatic densities. Note that there is no association between density on soma and density on dendrites (regression coefficient of dendrite on soma not significantly different from 0).

72

ROBERT F. SPENCER AND PETER STERLING TABLE 1

Synaptic density (synapsesi100 FmZ)and S/F ratios calculated from 3- 7sectaons through each of 18 motoneurones Cell no.

Diameter (pm)

1 24 2 27 3 29

4 32 5

33

6

36 37

37 9

38 10 40 11

43

12 44 13

44

14 44

Perimeter (&mi

59 77 48 64 92 58 71 102 67 43 85 135 101 98 140 105 90 104 100 120 133 140 141 128 143 95 45 148 138 145 155 120 85 105 155 121 75 99 123 133 144 166 158 138 133 125 111 110 116 153 145 153 106 164 140 133 131 125 123 161 148 148 135 120

S

F

7 17 10 5 7 6 10 16 6

6 7 6 7 6 4 8 12 7 4 8 14 5 11 14 8 23 8 14 19 20 22 17 7 6 5 2 8 12 15 18 12 8 10 16 15 4 9 14 22 17 23 20 27 26 21 19 17 9 16 32 30 18 35 21 28 25 21 17 33 28 17 21 22

6 13 10 6 12 15 10 4 11 5 14 7 10 18 8 8 8 2 26 10 11 13 17 3 8 9 14 11 5 15 3 13 16 17 9 14 13 13 15 22 17 10 12 17 15 30 18 13 13 16 14 6 16 20 14

Synapsed 100

4.8 9.6 11.0 3.5 2.0 2.9 6.4 7.5 3.7 5.4 6.1 3.2 1.2 5.6 4.3 2.9 8.9 3.3 3.6 7.6 4.2 5.2 6.1 1.4 0.9 1.9 0.8 5.3 2.6 3.2 4.0 5.8 1.7 2.9 2.6 5.7 4.0 2.0 5.6 3.5 4.3 5.5 5.5 6.9 9.1 7.4 8.2 8.5 7.1 4.7 8.4 7.6 10.8 9.3 13.3 12.0 8.4 7.4 7.2 8.5 5.3 5.0 9.1

9.0

SIF ratio

1.2 2.4 1.7 0.7 1.2 1.5 1.3 1.3 0.8 1.5 1.6 0.7 1.2 1.1 1.1 1.2 0.2 1.4 0.4 0.7 0.4 0.5 1.1 1.1 1.3 1.6 1.0 3.3 0.8 0.7 0.7 1.4 0.4 0.8 0.7 0.9 2.7 0.6 1.1 0.1 0.8 0.7 0.8 0.3 0.5 0.6 0.7 0.9 2.4 1.1 0.3 0.4 0.9 0.4 1.4 0.6 0.5 0.6 0.9 0.4 0.2 0.9 0.9 0.6

73

ABDUCENS MOTONEURONES A N D I N T E R N E U R O N E S TABLE 1 lrontinued)

Synaptic density (synapsed100 @m2)and S / F ratios calculated from 3-7sections through each of 18 motoneurones Diameter

Cell no

(Fml

15 49

16

49

17 49 18

59

Perimeter ~&rn)

114 138 145 149 133 110 181 156 121 140 193 153 84 200 230 133 123 81

S

F

21 14 16 17 22

15 23 20 23 19 18 20 20 29 11 36 21 11 20 16 21 25 19

8 17 9 6 24 14 12

7 28 37 7

9 17

Distribution of boutons containing spheroidal of flattened synaptic vesicles In single sections through the nucleolar plane of 121 motoneurones and 34 interneurones, the distribution of boutons containing either spheroidal or flattened synaptic vesicles was examined, since this may reflect the distribution of excitatory and inhibitory synaptic endings over the soma-dendritic surface (Uchizono, '65, '66). We were interested to learn whether the ratios of boutons containing spheroidal synaptic vesicles to those containing flattened vesicles (S/F ratio) were different for the two populations of neurones and also whether there were differences between cells within the motoneurone population. As shown in figure 5, the distributions of S/F ratios for the motoneurone and interneurone populations were virtually identical. I t proved impossible, however, to determine from single sections whether there were real differences in S/F ratios between motoneurones because the standard deviation of the mean was so large (1.4 2 1.0).The reason for this large variation was that boutons with similar vesicle morphology were frequently arranged in irregular homogeneous clusters on the somatic surface; consequently, any given section could be quite unrepresentative of the S/F ratio of the cell a s a whole. When the S/F ratios were studied by pooling data from three to seven sections through each of 18 motoneurones (table 11, the mean ratio for all cells was 1.0 f 0.3. As shown in

Synapses/ 100 p m 2

10.0 7.2 6.1 7.2 9.6 5.6 4.2 3.4 8.3 6.3 6.7 4.7 4.6 5.8 5.3 4.8 7.7 19.5

S/F ratio

1.4 0.6 0.8 0.7 1.2 0.4 0.8 0.4 0.2 2.2 0.4 0.6 0.6 1.4 2.3 0.3 0.4 0.9

figure 6, in no case but one (cell 1) did the mean ratio for an individual cell differ significantly from 1.0. It appears, therefore, that this parameter is quite constant from motoneurone to motoneurone, in contrast to the highly significant variation between cells in the overall density with which boutons are distributed on the somatic surface. The clustering of boutons containing either spheroidal or flattened synaptic vesicles, while quite irregular, proved to be non-random when analyzed statistically (one-sample runs test and Chi square distribution, p < 0.05). The S/F ratios on the dendrites of 16 motoneurones studied in detail were, on the average, significantly higher than those on the somata. Apparently, there are not only more boutons per unit area on the dendrites, but the boutons also more frequently contain spheroidal synaptic vesicles. There was also a clearly discernible pattern to the differences in S/F ratio between dendrites on the same motoneurone. As shown in figure 7, laterallyoriented dendrites almost invariably had lower S/F ratios than other dendrites of different orientation emanating from the same cell. The S/F ratios of laterally-oriented dendrites, with two exceptions (cells 15 and 18), were less than 1.0; in other words, the lateral dendrites had a relatively high proportion of synaptic endings containing flattened synaptic vesicles. This may be related to the fact that these dendrites are directed toward the ipsilateral medial vestibular nucleus,

74

ROBERT F. SPENCER AND PETER STERLING 5.0 1

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NEURONE DIAMETER Fig. 5 Distribution of S/F ratios of axosomatic boutons determined from single sections through nucleolar plane of 121 motoneurones (MN) and 34 interneurones (IN). Note that distribution of S/F ratios of both cell types overlap extensively.

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which is a recognized source of inhibitory input to abducens motoneurones (Precht and Richter, '68; Precht et al., '69; Baker et al., '69; Maeda et al., '71, '72; Highstein, '73). DISCUSSION

I t is clear that the cat abducens nucleus contains a rather heterogeneous population of neurones. These include motoneurones innervating the lateral rectus muscle, motoneurones innervating the retractor bulbi muscle, and interneurones, possibly of two types, pro-

jecting to the oculomotor nucleus. We had hoped to find criteria by which abducens motoneurones could be distingguished in normal material. Unfortunately, their properties overlap in most respects. Thus, their distributions within the nucleus, while not identical, do not permit their distinction based on location. The size distributions of the motoneurones and interneurones overlap completely, and neither their cytology nor their synaptology studied a t the ultrastructural level are distinctive. Although we did not study the dis-

75

ABDUCENS MOTONEURONES AND INTERNEURONES

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2.0

SOMA S/F RATIO Fig. 7 Plot of S/F ratios on dendrites of different orientation derived from 16 motoneurones studied in three to seven sections. All points below and to right of dotted line represent dendritic S/F ratios less than respective somatic S/Fratios. Note t h a t with only one exception (cell 18,arrow), S/Fratios of laterally-oriented dendrites are less than S/F ratios of dendrites of other orientations on same cell. Note also that S/F ratios of laterally-oriented dendrites, with two exceptions (cells 15 and 18, arrows), are all less than 1.0.

tribution of boutons on the interneurones as carefully as for the motoneurones, it is clear that no simple counting procedure can distinguish the two classes of cells in single sections. One subtype of interneurone seems to be distinguished from both motoneurones and other interneurones by its fusiform cell body, poorly developed granular endoplasmic reticulum, and fluted or invaginated nuclear envelope. If these distinctions are supported by experimental manipulations, they may prove useful in identifying this subtype. I t is clear, however, that in future studies of the patterns formed by specific afferents to the nucleus, i t will be necessary to separately identify the motoneurones and interneurones experimentally, as we have done in the present study. Our efforts to estimate the relative numbers of neurones belonging to each population

produced a paradoxical result: estimates based on individual experiments total more than 100%.Thus, in separate experiments, the lateral rectus motoneurones account for 85% of the neurones in the nucleus, the interneurones for 25%, and the retractor bulbi motoneurones for 30% a t the mid-level of the nucleus. These figures are estimates based on a systematic sampling of the nucleus, so it is possible that some of these obvious discrepancies could result from sampling errors. However, since the distributions of lateral rectus motoneurones and interneurones were rather uniform throughout the nucleus, it seems unlikely that this source could account for a significant degree of variation. It is also possible that there may be individual differences between animals in the relative numbers of each population of neurones, though it would be surprising if these were large enough to

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ROBERT F. SPENCER AND PETER STERLING

account for such a discrepancy. The relative number of lateral rectus motoneurones would be overestimated if HRP from injections into t h e lateral rectus muscle diffused within t h e orbit to the retractor bulbi muscle. Care was taken during the injection, however, to prevent such diffusion. That no labelled cells were found in the trochlear or oculomotor nuclei suggests t h a t these measures were effective. A more interesting possibility is t h a t some of the neurones in the abducens nucleus innervate more t h a n one effector structure. For example, if some of t h e motoneurones sent axon collaterals to the oculomotor nucleus, they would be labelled by HRF injections into the oculomotor nucleus and counted as “interneurones.” This could explain why t h e independent estimates of the percentages of lateral rectus motoneurones and abducens interneurones total more than 100%.I t could, furthermore, explain why one of the two morphological types of interneurones so closely resembles the motoneurones cytologically. Although, at present, anatomical (Ramon y Cajal, ’09; Lorente de No, ’33) and physiological (Baker et al., ’69; Baker and Highstein, ’75) observations suggest t h a t abducens motoneurones do not have axon collaterals, our findings suggest t h a t this possibility should be considered.

Synaptic patterns The distribution of boutons over t h e somadendritic surfaces of the abducens motoneurones seems to be governed by certain broad rules. First, for all motoneurones, there is a rather constant ratio of boutons containing spheroidal synaptic vesicles to those containing flattened vesicles. This ratio is approximately 1.0 for the cell somata and somewhat greater (2.3) for t h e dendrites. Apparently, there is relatively greater inhibitory input to t h e motoneurones soma t h a n to the proximal dendrites, with the possible exception of t h e lateral dendrites. The S/F ratio for t h e motoneurone somata in t h e abducens nucleus is slightly higher t h a n what we have calculated for the spinal cord from Conradi’s (’69) data and from the d a t a of Kojima e t al. (’72). The relative paucity on abducens motoneurone somata of boutons containing flattened vesicles may reflect the lack of inhibitory feedbacks from Renshaw, and Ia and Ib inhibitory interneurones. This may in t u r n partly explain the undamped firing of ab-

ducens motoneurones, the frequency of which reaches as much as ten times t h a t of spinal motoneurones (Eccles et al., ’58; Precht e t al., ’67; Baker et al., ’71). The absence of axoaxonic synapses in t h e abducens nucleus and their presence in the spinal motor nuclei may be related to the same phenomenon. The second rule is t h a t the boutons containing either spheroidal or flattened vesicles are distributed neither in a random pattern over the motoneurone surface nor in a regular, “checkerboard” array, but in relatively homogeneous clusters. This is consistent with our observations on single sections and is t h e only way one can account for the significant variation in SIF ratio between sections taken a t different levels through the same cell (table 1).I t is impossible to determine in the present material whether more than one afferent source contributes to each cluster. I t will be necessary to answer this question before one can make use of t h e anatomical data in e s t i m a t i n g t h e relative sizes of c u r r e n t sources and sinks over the cell surface. The third rule t h a t emerged in t h e present study is t h a t t h e total density of boutons is higher on t h e motoneurone dendrites t h a n on t h e somata. This was true not only on the average, but also for comparisons of individual cells. This rule may reflect a broad compensation for the attenuation of excitatory voltages in dendrites. Alternatively, the rule may have a more specific meaning t h a t will be clear only when the distribution of specific afferent sources has been studied. Finally, we found t h a t t h e total density of boutons varied significantly between motoneurones even though the ratio of boutons with spheroidal and flattened vesicles was constant. The range of bouton densities was similar to t h a t reported for spinal motoneurones (Conradi, ’69; Kojima e t al., ’721, but was substantially less t h a n t h e densities determined directly by Gelfan and Rapisarda (’64). The variation in total synaptic density between cells was not correlated with t h e diameter of the motoneurone soma, location within the abducens nucleus, nor with any other parameter t h a t we could think of. Again, the meaning of such large differences cannot be established i n the absence of information about t h e distribution of specific afferents. In summary, there appear to be a number of rules t h a t govern the distribution of boutons on t h e dendrites and somata of motoneurones.

ABDUCENS MOTONEURONES AND INTERNEURONES

There are some relatively constant features shared by all the cells, e.g., the somatic S/F ratio is approximately 1.0. Other features, such as total density of boutons and dendritic S/F ratio, seem more tailored for each motoneurone. This is not surprising in view of the known physiological differences between abducens motor units (Lennerstrand, '75; Goldberg e t al., '76). The present study has served primarily to define some guidelines and questions that should be useful in investigating a t the ultrastructural level the distribution of specific afferents within the abducens nucleus. LITERATURE CITED Alvarado, J . A., and C. Van Horn 1975 Muscle cell types in the cat inferior oblique. In: Basic Mechanisms of Ocular Motility and t h e i r Clinical Implications. G . Lennerstrand and P . Bach y Rita, eds. Pergamon Press, Oxford, pp. 15-43. Bach y Rita, P. 1971 Neurophysiology of eye movements. In: The Control of Eye Movements. P. Bach y Rita and C. C. Collins, eds. Academic Press, New York, pp. 7-45. Bach y Rita, P., and F. Ito 1965 In U I U O microelectrode studies of the cat retractor hulbi fibers. Invest. Ophthal., 4: 338-342. Baker, R., and S. M. Highstein 1975 Physiological identification of interneurones and motoneurones in the ahducens nucleus. Brain Res., 91: 292-298. Baker, R. G., N. Mano and H. Shimazu 1969 Postsynaptic potentials in ahducens motoneurones induced by vestihular stimulation. Brain Res., 25: 577-580. Bodian, D. 1970 An electron microscopic characterization of classes of synaptic vesicles by means of controlled aldehyde fixation. J . Cell Biol., 44: 115-124. Brightman, M. W., and T. S. Reese 1969 Junctions between intimately a p p o s d cell membranes in the verte brate brain. d. Cell Biol., 40: 648-677. Buttner-Ennever, J. A., and V. Henn 1976 An autoradiographic study of t h e pathways from t h e pontine reticular formation involved in horizontal eye movements. Brain Res., 208: 155.164. Colonnier, M. 1968 Synaptic patterns on different cell types in t h e different laminae of t h e cat visual cortex. An electron microscope study. Brain Res., 9: 268-287. Conradi, S. 1969 Ultrastructure and distribution of neuronal and glial elements on t h e motoneurone surface in the lumbosacral spinal cord of t h e adult cat. Acta Physiol. Scand. Suppl., 332: 5-48. Eccles, J. C., R. M. Eccles and A. Lundberg 1958 The action potentials of t h e alpha motoneurones supplying fast and slow muscles. J. Physiol., 242: 275-291. Edwards, S. B. 1972 The ascending and descending projections of t h e red nucleus i n the cat: a n experimental study using a n autoradiographic tracing method. Brain Res., 48: 45-63. Fuchs, A. F., and E. S. Luschei 1971 Development of isometric tension in simian extraocular muscle. J . Physiol., 219: 155-166. Fuse, G. 1912 Uber den Ahduzenskern der Sauger. Arb. Hirnanat. Inst. Zurich, 6: 401-447. Gacek, R. R. 1971 Anatomical demonstration of t h e vestihulo-ocular projections in the cat. Acta oto-laryng. (Stockholm), (Suppl.), 293: 1-63.

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1974 Localization of neurons supplying the extraocular muscles in the kitten using horseradish per ox^ idase. Exptl. Neurol.. 44: 381-403. Gelfan, S., and A. F. Rapisarda 1964 Synaptic density on spinal neurons of normal dogs and dogs with experimental hind~limhrigidity J. Comp. Neur., 123: 73-95. Gogan, P., J. P. Gueritaud, G. Horcholle~Bossavitand S. Tyc-Dumont 1974 Electrotonic coupling between motoneurones in the ahducens nucleus of the cat. Exptl. Brain Res., 21: 139-154. Goldberg, S. J . , G. Lennerstrand and C. D. Hull 1976 Motor unit responses in the lateral rectus muscle of the cat: intracellular current injections of abducens nucleus neurons. Acta Physiol. Scand., 96: 53-63. Graham, R. C., Jr., and M. J . Karnovsky 1966 The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem., 14: 291-302. Graybiel, A. M. 1975 Anatomical pathways in t h e hrainstem oculomotor system. In: Eye Movements and Motion Perception. Ninth Symposium of t h e Center for Visual Science, Rochester, N. Y.. pp. 37-38. Graybiel, A. M., and E. A. Hartwieg 1974 Some afferent connections of t h e oculomotor complex in the c a t : a n experimental study with tracer techniques. Brain Res., 81: 543-551. Henn. V., and B. &hen 1972 Eye muscle motor neurons with different functional characteristics. Brain Res., 45: 561- 568. Highstein, S. M. 1973 Synaptic linkage in the vestihuloocular and cerebello-vestibular pathways t o the Vlth nucleus in t h e rabbit. Exptl. Brain Res., 17: 301-314. Highstein, S. M., K. Maekawa, A. Steinacker and B. Cohen 1976 Synaptic input from the pontine reticular nuclei to abducens motoneurons and internuclear neurons in the cat. Brain Res., 112: 162-167. Karnovsky, M. J. 1967 The ultrastructural basis of capillary permeability studied with peroxidase a s a tracer. J. Cell Biol., 35: 213-236. Keller, E. L., and D. A. Robinson 1971 Absence of a stretch reflex in extraocular muscles of t h e monkey. J . Neurophysiol., 34: 908-919. ___ 1972 Ahducens unit behavior in the monkey during vergence movements. Vision Res., 12: 369-382. Kojima, T., K. Saito and S. Kakimi 1972 Electron microscopic quantitative observations on the neuron and the terminal boutons contacted with it in t h e ventrolateral part of t h e anterior horn (C, ,I of the adult cat. Okajimas Folia a n a t . jap., 49: 175-226. Kristensson, K., Y. Olsson and J . Sjostrand 1971 Axonal uptake and retrograde transport of exogenous proteins in t h e hypoglossal nerve. Brain Res., 32: 222-231. La Vail, J . H., and M. M. La Vail 1972 Retrograde axonal transport in t h e central nervous system. Science, 176: 1416-1417. 1974 The retrograde intraaxonal transport of horseradish peroxidase in the chick visual system: a light and electron microscope study. J . Comp. Neur.. 257: 303-358. Lennerstrand, G. 1975 Motor units in eye muscles. In: Basic Mechanisms of Ocular Motility and their Clinical Implications. G. Lennerstrand and P. Bach y Rita, eds. Pergamon Press, Oxford, pp. 119-143. Lorente de No, R. 1933 Vestibule-ocular reflex arc. Arch. Neurol. Psychiat., 30: 245-291. Maciewicz, R. J., C. R. S. Kaneko, S. M. Highstein and R. Baker 1975 Morphophysiological identification of interneurones in t h e oculomotor nucleus t h a t project to the abducens nucleus in the cat. Brain Res., 96: 60-65. ~

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Maeda. M.. H. Shimazu and Y. Shinoda 1971 Inhibitory postsynaptic potentials in the abducens motoneurones associated with t h e quick relaxation phase of vestibular nystagmus. Brain Res., 26: 420-424. 1972 Nature of synaptic events in c a t abducens rnotoneurons a t slow and quick phase of vestibular nystagmus. J. Neurophysiol., 35: 279-296. McCouch. G. P.. and F. H. Adler 1932 Extraocular reflexes. Am. J . Physiol.. 100; 78-88. McMasters. R. E.. A H. Weiss and M. B. Carpenter 1966 Vestibular projections to the nuclei of the extraocular muscles. degeneration resulting from discrete partial lesions of the vestibular nuclei in t h e monkey. Am. J. A n a t , 118: 163-194. Paula-Barbosa. M. 1975 The duration of aldehyde fixation a s a "flattening factor" of synaptic vesicles. Cell Tiss Res.. 164: 63-72. Peachey. L. 1971 The structure of the extraocular muscle fibers of mammals. In: The Control of Eye Movements. P. Bach y Rita and C. C. Collins, eds. Academic Press. New York, pp. 47-66. Precht. W., J . Grippo and A. Richter 1967 Effect of horizontal angular acceleration on neurons in t h e abducens nucleus. Brain Res.. 5: 527-531. Precht. W.. A. Richter and J. Grippo 1969 Responses of neurones in the cat's abducens nuclei to horizontal angular acceleration. Pflugers Arch. ges. Physiol., 309: 285309. Ramon y Cajal. S. 1909 Histologie du Systeme Nerveux de 1'Homrne et des Vertebres. Tome I. (reprinted 1972). C.S.I.C.. Madrid, pp 854-858. Reynolds, E. S. 1963 The use of lead citrate a t high pH as an electron-opaque stain for electron microscopy. J. Cell Biol.. 17. 208-212. Richter, A,, and W. Precht 1968 Inhibition of abducens motoneurons by vestibular nerve stimulation. Brain Res.. 11: 701-705. Robinson. D. A. 1970 Oculomotor unit behavior in t h e monkey. J. Neurophysiol., 38: 393-402. Romanes, G. J. 1951 The motor cell columns of t h e lumbrosacral spinal cord of the cat. J . Comp. Neur.. 94: 313-364. Saito. K. 1972 Electron microscopic observations on t e r minal boutons and synaptic structures in t h e anterior

horn of the spinal cord of t h e adult cat. Okajimas Folia anat. jap., 48: 361-412. Schaefer, K. P . 1965 Die Erregungsmuster einzelner Neurone d e s Abducens-Kernes beim K a n i n c h e n . Pflugers Arch. ges. Physiol., 284: 31-52. Schiller, P. M. 1970 The discharge characteristics of single units in t h e oculomotor and abducens nuclei of the unanesthetized monkey. Exp. Brain Res., 20: 347-363. Sotelo, C., R. Linas and R. Baker 1974 Structural study of inferior olivary nucleus of t h e cat: morphological correlates of electronic coupling. J . Neurophysiol., 37: 541559. Sterling, P. 1977 Anatomy and physiology of t h e goldfish oculomotor system. I. Structure of the abducens nucleus. J . Neurophysiol., 40: 557-572. Sterling, P.. and H. G. J. M. Kuypers 1967 Anatomical organization of the brachial spinal cord of t h e cat. 11. The motoneuron plexus. Brain Res., 4: 16-32. Szentagothai, J . 1950 The elementary vestibule-ocular reflex arc. J . Neurophysiol., 13: 395-407. Taber, E. 1961 The cytoarchitecture of the brain stem of the cat. I. Brain stem nuclei of the cat. J . Comp. Neur., 116: 27-69. Tarlov, E. 1970 Organization of vestibulo-oculornotor projections in the cat. Brain Res., 20: 159-179. Uchizono. K. 1965 Characteristics of excitatory and inhibitory synapses i n the central nervous system of the cat. Nature, 207: 642-643. 1966 Excitatory and inhibitory synapses in the cat spinal cord. Jap. J. Physiol., 16: 570-575. Valdivia, 0. 1971 Methods of fixation and t h e morphology of synaptic vesicles. J. Comp Neur., 142: 257-274. van Gehuchten, A. 1898 Recherches sur I'Origine r k l e des Nerfs craniens: I. Les Nerfs moteur oculaires. J . Neurol. Hypnol., 3: 114-129. Walberg, F. 1966 Elongated vesicles in terminal boutons of the central nervous system, a result of aldehyde fixation. Acta Anat.. 65: 224-235. Waxman. S. G.. and G. D. Pappas 1971 Synaptic organization of t h e oculomotor nucleus: a comparative electron microscopic study. Biol. Bull., 139. 442. Yamanaka, Y . , and P. Bach y Rita 1968 Conduction velocities in t h e abducens nerve correlated with vestibular nystagmus in cats. Exptl. Neurol., 20: 143-155.

PLATES

PLATE 1 EXPLANATION 01.' FIGURES

8

Light micrograph idarkfield illumination) of semithin section through abducens nucleus showing HRP-labelled motoneurones. X 200.

9 Low magnification electron micrograph of ultrathin section adjacent to semithin section shown in figure 8. X 200. 10 Electron micrograph of HRP-labelled abducens motoneurone (outlined cell in figs. 8 and 9 ) .Morphological features characteristic of all abducens motoneurones include a circular nucleus with a smooth membrane and well developed cisternal arrays of granular endoplasmic reticulum. Boutons containing spheroidal synaptic vesicles are indicated by outlined arrows and boutons containing flattened vesicles are indicated by solid arrows. X 2,975

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ABDUCENS MOTONEURONES A N D I N T E R N E U R O N E S Robert F Spencer and Peter Sterling

PLATE 1

81

PLATE 2 EXPLANATION OF FIGURES

11 Light micrograph of semithin section through abducens nucleus showing HRP-labelled interneurones. indicated by arrows. Note unlabelled neurones, indicated by asterisks (*). X 280. 12 Electron micrograph of fusiform interneurone. Note poorly developed granular endoplasmic reticulum and deeply invaginated nuclear membrane, features characteristic of this type of interneurone. Boutons containing spheroidal synaptic vesicles are indicated by outlined arrows and boutons containing flattened vesicles are indicated by solid arrows. X 2,335. 13 Electron micrograph of interneurone containing well developed cisternae of granular endoplasmic reticulum and circular nucleus with smooth nuclear membrane. Boutons containing spheroidal synaptic vesicles are indicated by outlined arrows and boutons containing flattened vesicles are indicated by solid arrows. X 2,335.

82

PLATE 3 EXPLANATION O F FIGURES 14

Electron micrograph of axodendritic boutons containing either spheroidal (S) or flattened (F) synaptic vesicles. Arrows indicate synaptic contact zones. Incubation for HRP reaction product produced no obvious alterations in morphology of synaptic vesicles. X 40,700.

15

Electron micrograph of a flattened vesicle bouton making multiple synaptic contacts (arrows1 on a motoneurone soma. Note two smaller boutons, also containing flattened synaptic vesicles, but differing in other axoplasmic organelles, on either side of the large bouton. X 7,335.

16 Electron micrograph of desmosome-like apposition between primary dendrite (Dl and soma (S) of 2 HRP-labelled motoneurones. Apposed membranes (arrows) are sep-

arated by cleft of 180 A, with flocculent electron-dense material aggregated along and between pre- and postjunctional membranes. X 33,335.

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ABDUCENS MOTONEURONES AND INTERNEURONES Robert F Spencer and Peter Sterling

PLATE 3

85