- Email: [email protected]
Relationship between motoneurons and internuclear neurons in the abducens nucleus: a double retrograde tracer study in the cat
Brain Research, 148 (1978) 181-188 © Elsevier/North-HollandBiomedical Press
181
Short Communications
Relationship between motoneurons ,and internuclear neurons in the abducens nucleus: a double retrograde tracer study in the cat H.-J. STEIGER and J. BOTTNER-ENNEVER Brain Research Institute, University of Ziirich, 8029 Ziirich, (Switzerland)
(Accepted December 8th, 1977)
Recent electrophysiological experiments provide strong evidence for the existence of neurones in the abducens nucleus which do not send axons to the lateral rectus muscle 1A7. The cells projecting instead to the oculomotor nucleus have been called internuclear neuronesl, z°. In anatomical studies on the rabbit 14, cat 14,19, and monkey s-l° the axons of the internuclear neurons have been shown to cross the midline, ascend in the medial longitudinal fasciculus and terminate exclusively in the oculomotor nucleus s,19. This pathway is essential for adduction of the eye in horizontal conjugate gaze a-5,7,9-12,19. Fuse 14 demonstrated the existence of these internuclear neurones in 1912, but the relative location and proportion of motoneurons to internuclear neurons are still open questions. There are conflicting reports on the number of abducens neurons undergoing chromatolysis after severing the VI th nerve: Warwick 25 and Carpenter et al. 9 found retrograde changes in nearly all the cells in monkey, while Fuse 14, in both cat and rabbit, reported chromatolysis in only a limited number of cells. Gacek 15 identified the motoneurones by injecting horseradish peroxidase (HRP) into the lateral rectus of kittens and found labelling ranged from less than 50 ~-90 ~ of the population. In addition there is no estimate of the number of neurons that project to the oculomotor nucleus in the catl4,19 and an insignificant percentage was found to undergo chromatolysis after cutting the medial longitudinal fasciculus in the monkey 10. The lack of clear cytological evidence for these two separate cell groups has led to the suggestion that the projection to the oculomotor nucleus could arise from collaterals of the motoneurones, although Golgi studies have failed to reveal them 24. It is interesting that a recent study on the ultrastructure of cat abducens neurones has revealed multiple branching of the axons 13. In the present study the relative proportion and the localization of the two cell types were studied, and the collateral hypothesis tested, by simultaneous retrograde labelling of the two populations with two different tracer substances, Evans blue (EB) and horseradish peroxidase (HRP). Five cats, not older than 6 months, were anaesthetized with 35 mg/kg sodium
182 pentobarbital and 4/zl of a 40 ~o Evans brae solution (Merck) zz were injected stereotaxically into the oculomotor complex 6. Using the same principle as Gacek 15, the right lateral rectus muscle was exposed by a lateral approach, and 50/zl of a 50 o/,,, horseradish peroxidase solution (Sigma type VI) were injected slowly into the muscle. After a survival time of 2 days the animals were reanaesthetized and perfused with a phosphate buffered solution (pH 7.4) containing 2.5~o glutaraldehyde and 0.4~o paraformaldehyde. The brains were removed and left in fixative overnight, then transferred to a 0.1 M phosphate buffer solution (pH 7.4) containing 30°/,i sucrose. Frozen sections (40 #m) were taken in the transverse plane 6 and sections from the abducens nucleus were stained for H R P 18. Following incubation for 10 min in a Tris buffered solution (pH 7.6) containing 0.06 ~o diamino-benzidine-tetrahydrochloride (Fluka) and 0.01 ~ H202 the sections were mounted and air-dried quickly to prevent loss of EB. Clearing the sections was reduced to a 5 min treatment in xylol. The sections were studied under light-field illumination at magnifications up to × 440. For the morphometric study the exact positions of EB- and HRP-labelled cells of every third section were charted using an X - Y plotter coupled to the microscope. Finally, these sections were counterstained with cresyl violet to compare the numbers of labelled cells with the total neuronal population in abducens nucleus. The number of labelled neurones and the total cell number were extrapolated from the sample. A correction was made according to the ratio between the number of perikarya and the number of nucleoli counted in the abducens nucleus for each experiment 21. The spatial relationships of E B - a n d HRP-labelled cells including density ratios 2 were calculated by a PDP-I 1 computer. EB-labelled cells in the abducens nucleus are characterized by heavily stained blue granules of approximately 1.2 #m in diameter, which are associated with the cell nucleus, while the cytoplasm contains finer blue granules superimposed on a homogeneous blue background (Fig. lc and d). Motoneurons labelled with H R P are filled with fine brown granules; often the cytoplasmic background appears weakly brown, but no granules are associated with the nucleus (Fig. lb). The injection sites of cases 77-259, 77-280 and 77-281 are shown in Fig. l a, and in these experiments there was successful labelling of the right abducens nucleus with both EB and HRP. Although the injection sites were very similar in 4 cases the intensity of EB in the abducens neurons varied considerably. There was a similar variation in the uptake of H R P from the lateral rectus muscle 15. All the sections of the successful cases were TABLE I Labelling o f abducens neurons in the cat
Number of HRP labelled motoneurons Number of EB labelled internuclear neurons Number of double-labelled neurons Total nm of cells in abducens nucleus recounted after Nissl staining
Exp. 77-280
77-281
1216 594 none 2067
1152 555 none 1824
183
77-259
77-280
77-281 a
Fig. 1. a: the injection sites of 3 experiments in the oculomotor nucleus (II1) at 3 different rostrocaudal levels. The heavily blue stained central area, where EB uptake is thought to take place is shaded. The broken lines identify the outer limits of the visible diffusion. This latter zone is not considered to be effective in EB uptake. MLF, medial longitudinal fasciculus; p, pontine nuclei; rn, red nucleus; IV, trochlear nucleus, b: motoneuron of VI th nerve nucleus identified by retrograde H R P labelling. c: internuclear neurons of VI th nerve nucleus identified by retrograde EB labelling. Note the darkly stained structures associated with the EB-labelled cell nucleus marked by an arrow, and shown at a higher magnification in d. This feature was important in detecting the more weakly EB-labelled cells. The brown and blue colour differences which chiefly characterise the two tracer substances are not visible here.
184 ROSTRAL 3
m 5
•
II
m
? m
c,,uoA,
77 ,
-
280
I mm
n •
HRP Evans-Blue
m Figs. 2 and 3. Drawings of every third section of the right abducens nucleus in experiment 77-280 and 77-281. Black dots, EB-labelled internuclear neurons projecting to the contralateral oculomotor nucleus. Triangles, abducens motoneurons labelled with HRP. N VII, knee of facial nerve; m, midline.
examined for double-labelled neurones. However, we encountered only two distinct types of neurones, containing either H R P or EB, and no double-labelled cells were found. This is further evidence against the hypothesis that the internuclear fibers are abducens axon collaterals, especially since the efficiency of labelling in cases 77-280 and 77-281 was so high. Cell counts were performed in these two cases and the labelling of the two cell pools is reflected in the respective totals shown in Table 1. Almost the total number of abducens neurones is accounted for by the sum of the motoneurones and internuclear neurons. A small difference (not exceeding 10 %) remains and could represent a second class of interneurons projecting to another target area. Unfortunately, we were not able to obtain direct evidence because the unstained cells
185
i,,
I,
~'~
o~a
"A~
I I m
~
•
ITI 10 ~
11
.
rn
~_j~ m
~ I, 1 r a m
j
77-281 •
HRP Evons-Blue
Fig. 3. which failed to be labelled by H R P and EB markers were not identifiable, for technical reasons. The ratio of motoneurones/internuclear neurones in experiments 77-280 and 77-281 is 2:1 (Table I). The distribution of the two populations of cells was similar in all cases; both types of cells could be found throughout the whole nucleus; that is the internuclear cell pool was confined to the boundaries of the abducens nucleus, as defined by the area enclosing the motoneurons. However, in all experiments the most rostral sections contained more internuclear neurons while motoneurons predominated in the caudal parts of the nucleus (Figs. 2 and 3). This is in good agreement with physiological data 1. Computer analysis proved useful in clarifying the spatial arrangement of the cells, especially the clustering of the two types. The ratio of internuclear neurones/
186
~ ~o c
~,
~ 08
.~ 2s
0.6
~o
o
i ~ 02 o
~
o
2;0 Distance (/urn)
3'0
2;0 Distance
(/urn)
Fig. 4. Computer analysis showing the tendency of motoneurons and internuclear neurons to form separate clusters. The left side is the average value of the motoneuron/internuclear neuron ratio, plotted against the distance from a motoneuron; the right side represents the average ratio of internuclear to motoneurons plotted against the distance from an internuclear neuron. Note the decrease in the ratios with increasing distance, which reflects the formation of separate clusters. Open circles 77-280, filled circles 77-281. motoneurones in the vicinity of any given internuclear neuron drops from a relatively high value to the ratio of the two labelled cell pools within the abducens nucleus. It can be seen in Fig. 4 that within a distance of 120/am from an internuclear neuron the average ratio of internuclear neurons/motoneurons is 1.7 times higher than the overall ratio. The same is true for motoneurones. Within 120/~m from a motoneurone the ratio of motoneurones/internuclear neurones is on average 1.4 times higher than the motoneurones/internuclear neurone ratio for the whole nucleus. Thus, the tendency for the two types of cells to form separate clusters is clearly established. In spite of the successful use of Evans blue in these experiments, this dye is not a consistently sensitive retrograde tracer substance. It gave reasonably clear results when injected into the oculomotor nucleus but proved to be unsuitable for labelling the motoneurons from the lateral rectus muscle. An important advantage of EB is that axon terminals are not labelled by anterograde transport, which, in a reciprocal system such as the interconnections between abducens and oculomotor nucleus 23, might obscure the identification of double-labelled neurones by light microscopy. Preliminary experiments with [aH]HRP as second tracer 16 failed partly as a result of this. Radioactive [3H]HRP appeared to label axon terminals heavily in the abducens nucleus by anterograde transport, which obscured autoradiographic analysis. In addition there was a poor retrograde transport of the radioactivity and a relatively high enzymatic activity. The detection of weakly labelled EB neurones was highly simplified by the characteristic staining of peri- or intranuclear structures (Fig. lc and d). This phenomenon may not be present in other systems: for example vestibular neurones that were labelled in the same experiment did not show similar dark blue grains associated with the cell nucleus. In the control experiment both H R P and EB were injected into the oculomotor nucleus at a slightly different rostrocaudal localization. A small number of double-labelled neurones were found within the vestibular complex. These neurones contained distinctly blue as well as greenish brown granules.
187 The techniques used in this study have p r o v i d e d a u n i q u e o p p o r t u n i t y to establish the location, p r o p o r t i o n (2:1) a n d spatial relation o f the m o t o n e u r o n e s a n d internuclear neurones in the a b d u c e n s nucleus o f the cat. I n a d d i t i o n they p r o v i d e further evidence against the collateral hypothesis. T h e a u t h o r s gratefully a c k n o w l e d g e the e n c o u r a g e m e n t o f Prof. K. A k e r t , the valuable help o f Dr. P. Streit who developed the m e t h o d o f r e t r o g r a d e tracing with Evans blue, a n d the technical assistance b y I. G y a r m a t i , A. F~ih, D. Savini a n d R. Emch. The w o r k was s u p p o r t e d by the Swiss N a t i o n a l Science F o u n d a t i o n , g r a n t N o . 3.636.75 a n d the Dr. Eric S l a c k - G y r F o u n d a t i o n in Zfirich.
1 Baker, R. and Highstein, S. M., Physiological identification of interneurons and motoneurons in the abducens nucleus, Brain Research, 91 (1975) 292-298. 2 Bendat, J. S. and Piersol, A. G., Random Data: Analysis and Mesaurement Procedures, John Wiley, New York, 1971, pp. 28-31. 3 Bender, M.B. and Weinstein, E. A., The syndrome of the median longitudinal fasciculus. In H. W. Woltman, H. H. Merritt, S. B. Wortis and C. C. Hare (Eds.), Multiple Sclerosis and the Demyelinating Diseases, Res. PubL Ass. nerv. ment. Dis., 28 (1950) 414-420. 4 Bender, M. B. and Shanzer, S., Oculomotor pathways defined by electrical stimulation and lesions in the brainstem of monkey. In M. B. Bender (Ed.) The Oculomotor System, Harper and Row, New York, 1964, pp. 81-140. 5 Bennett, A. H. and Savill, T., A case of permanent deviation of the eyes and head, the result of a lesion limited to the sixth nucleus, with remarks on associated lateral movements of the eye balls, and rotation of head and neck, Brain, 12 (1890) 102-116. 6 Berman, A. L., The Brain Stem of the Cat. A Cytoarchitectonic Atlas with Stereotaxic Coordinates, The University of Wisconsin Press, Madison, 1968, 175 pp. 7 Blocq, P. et Guignon, G., Paralysie conjugu6e de la sixi/:me paire, Arch. Med. exp., 3 (1891) 74-89. 8 B~ittner-Ennever, J. A. and Henn, V., An autoradiographic study of the pathways from the pontine reticular formation involved in horizontal eye movements, Brain Research, 108 (1976) 155-164. 9 Carpenter, M.B., McMasters, R.E. and Hanna, G. R., Disturbances of conjugate horizontal eye movements in the monkey. I. Physiological effects and anatomical degeneration from lesions of the abducens nucleus and nerve, Arch. Neurol. (Chic.), 8 (1963) 231-247. 10 Carpenter, M. B. and McMasters, R. E., Disturbances of conjugate horizontal eye movements in the monkey. II. Physiological effects and anatomical degeneration resulting from lesions in the medial longitudinal fasciculus, Arch. Neurol. (Chic.), 8 (1963) 347-368. 11 Cogan, D. C., Kubik, C. S. and Smith, W. L., Unilateral internuclear ophthalmoplegia, Arch. Ophthal., 44 (1950) 783-796. 12 Crosby, E. C., Relations of brain centers to normal and abnormal eye movements in the horizontal plane, J. comp. Neurol., 99 (1951) 437--480. 13 Destombes, J. and Ripert, J. P., Ultrastructurai observations of the abducens nucleus of the cat after injection of horseradish peroxidase into the lateral rectus muscle, Exp. Brain Res., 28 (1977) 63-71. 14 Fuse, G., Ueber den Abduzenskern der S/iuger. In Arbeiten aus dem Hirnanatomischen lnstitut der Universitiit Ziirich, VoL V1, Bergmann, Wiesbaden, 1912, pp. 401-447. 15 Gacek, R. R., Localization of neurons supplying the extraocular eye muscles in the kitten using horseradish peroxidase, Exp. NeuroL, 44 (1974) 381--403. 16 Geisert, E. E., Jr., The use of tritiated horseradish peroxidase for defining neuronal pathways: a new application, Brain Research, 117 (1976) 130-135. 17 Gogan, P., Gueritaud, J. P., Horcholle-Bossavit, G. and Ty6-Dttmont, S., Inhibitory nystagmic interneurons. Physiological and anatomical identification within the abducens nucleus, Brain Research, 59 (1973) 410-416. 18 Graham, R. C., Jr. and Karnovsky, M. J., The early stages of absorption of injected horseradish
188
19 20
21 22
23
24 25
peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. Graybiel, A. M. and Hartwieg, E. A., Some afferent connections of the oculomotor complex in the cat: an experimental study with tracer techniques, Brain Research, 81 (1974) 543-551. Highstein, S. M., Maekawa, K., Steinacker, A. and Cohen, B., Synaptic input from the pontine reticular nuclei to abducens motoneurons and internuclear neurons in the cat, Brain Research, 112 (1976) 162-167. Konigsmark, B. W., Methods for counting neurons. In W. J. H. Nauta and S. O. E. Ebbesson (Eds.) Contemporary Research, Methods in Neuroanatomy, Springer, Berlin, 1970, pp. 315-340. Kristensson, K., Retrograde axonal flow of protein tracers. In W. M. Cowan and M. Cu6nod (Eds.), The Use t~f Axonal Transport for Studies of Neuronal Connectivity, Elsevier, Amsterdam, 1975, pp. 69-82. Maciewicz, R. J., Kaneko, C. R. S., Highstein, S. M. and Baker, R., Morphophysiological identification of interneurons in the oculomotor nucleus that project to the abducens nucleus in the cat, Brain Research, 96 (1975) 60-65. Ram6n y Cajal, S., Histologie du Systdme Nerveux de l'Homme et des Vertebras, Vol. 1 (Azoulay translation), Institute Ram6n y Cajal, Madrid, 1955, pp. 856. Warwick, R., Oculomotor Organization. In M. B. Bender (Ed.) The Oculomotor System. Harper and Row, New York, 1964, pp. 173-204.
181
Short Communications
Relationship between motoneurons ,and internuclear neurons in the abducens nucleus: a double retrograde tracer study in the cat H.-J. STEIGER and J. BOTTNER-ENNEVER Brain Research Institute, University of Ziirich, 8029 Ziirich, (Switzerland)
(Accepted December 8th, 1977)
Recent electrophysiological experiments provide strong evidence for the existence of neurones in the abducens nucleus which do not send axons to the lateral rectus muscle 1A7. The cells projecting instead to the oculomotor nucleus have been called internuclear neuronesl, z°. In anatomical studies on the rabbit 14, cat 14,19, and monkey s-l° the axons of the internuclear neurons have been shown to cross the midline, ascend in the medial longitudinal fasciculus and terminate exclusively in the oculomotor nucleus s,19. This pathway is essential for adduction of the eye in horizontal conjugate gaze a-5,7,9-12,19. Fuse 14 demonstrated the existence of these internuclear neurones in 1912, but the relative location and proportion of motoneurons to internuclear neurons are still open questions. There are conflicting reports on the number of abducens neurons undergoing chromatolysis after severing the VI th nerve: Warwick 25 and Carpenter et al. 9 found retrograde changes in nearly all the cells in monkey, while Fuse 14, in both cat and rabbit, reported chromatolysis in only a limited number of cells. Gacek 15 identified the motoneurones by injecting horseradish peroxidase (HRP) into the lateral rectus of kittens and found labelling ranged from less than 50 ~-90 ~ of the population. In addition there is no estimate of the number of neurons that project to the oculomotor nucleus in the catl4,19 and an insignificant percentage was found to undergo chromatolysis after cutting the medial longitudinal fasciculus in the monkey 10. The lack of clear cytological evidence for these two separate cell groups has led to the suggestion that the projection to the oculomotor nucleus could arise from collaterals of the motoneurones, although Golgi studies have failed to reveal them 24. It is interesting that a recent study on the ultrastructure of cat abducens neurones has revealed multiple branching of the axons 13. In the present study the relative proportion and the localization of the two cell types were studied, and the collateral hypothesis tested, by simultaneous retrograde labelling of the two populations with two different tracer substances, Evans blue (EB) and horseradish peroxidase (HRP). Five cats, not older than 6 months, were anaesthetized with 35 mg/kg sodium
182 pentobarbital and 4/zl of a 40 ~o Evans brae solution (Merck) zz were injected stereotaxically into the oculomotor complex 6. Using the same principle as Gacek 15, the right lateral rectus muscle was exposed by a lateral approach, and 50/zl of a 50 o/,,, horseradish peroxidase solution (Sigma type VI) were injected slowly into the muscle. After a survival time of 2 days the animals were reanaesthetized and perfused with a phosphate buffered solution (pH 7.4) containing 2.5~o glutaraldehyde and 0.4~o paraformaldehyde. The brains were removed and left in fixative overnight, then transferred to a 0.1 M phosphate buffer solution (pH 7.4) containing 30°/,i sucrose. Frozen sections (40 #m) were taken in the transverse plane 6 and sections from the abducens nucleus were stained for H R P 18. Following incubation for 10 min in a Tris buffered solution (pH 7.6) containing 0.06 ~o diamino-benzidine-tetrahydrochloride (Fluka) and 0.01 ~ H202 the sections were mounted and air-dried quickly to prevent loss of EB. Clearing the sections was reduced to a 5 min treatment in xylol. The sections were studied under light-field illumination at magnifications up to × 440. For the morphometric study the exact positions of EB- and HRP-labelled cells of every third section were charted using an X - Y plotter coupled to the microscope. Finally, these sections were counterstained with cresyl violet to compare the numbers of labelled cells with the total neuronal population in abducens nucleus. The number of labelled neurones and the total cell number were extrapolated from the sample. A correction was made according to the ratio between the number of perikarya and the number of nucleoli counted in the abducens nucleus for each experiment 21. The spatial relationships of E B - a n d HRP-labelled cells including density ratios 2 were calculated by a PDP-I 1 computer. EB-labelled cells in the abducens nucleus are characterized by heavily stained blue granules of approximately 1.2 #m in diameter, which are associated with the cell nucleus, while the cytoplasm contains finer blue granules superimposed on a homogeneous blue background (Fig. lc and d). Motoneurons labelled with H R P are filled with fine brown granules; often the cytoplasmic background appears weakly brown, but no granules are associated with the nucleus (Fig. lb). The injection sites of cases 77-259, 77-280 and 77-281 are shown in Fig. l a, and in these experiments there was successful labelling of the right abducens nucleus with both EB and HRP. Although the injection sites were very similar in 4 cases the intensity of EB in the abducens neurons varied considerably. There was a similar variation in the uptake of H R P from the lateral rectus muscle 15. All the sections of the successful cases were TABLE I Labelling o f abducens neurons in the cat
Number of HRP labelled motoneurons Number of EB labelled internuclear neurons Number of double-labelled neurons Total nm of cells in abducens nucleus recounted after Nissl staining
Exp. 77-280
77-281
1216 594 none 2067
1152 555 none 1824
183
77-259
77-280
77-281 a
Fig. 1. a: the injection sites of 3 experiments in the oculomotor nucleus (II1) at 3 different rostrocaudal levels. The heavily blue stained central area, where EB uptake is thought to take place is shaded. The broken lines identify the outer limits of the visible diffusion. This latter zone is not considered to be effective in EB uptake. MLF, medial longitudinal fasciculus; p, pontine nuclei; rn, red nucleus; IV, trochlear nucleus, b: motoneuron of VI th nerve nucleus identified by retrograde H R P labelling. c: internuclear neurons of VI th nerve nucleus identified by retrograde EB labelling. Note the darkly stained structures associated with the EB-labelled cell nucleus marked by an arrow, and shown at a higher magnification in d. This feature was important in detecting the more weakly EB-labelled cells. The brown and blue colour differences which chiefly characterise the two tracer substances are not visible here.
184 ROSTRAL 3
m 5
•
II
m
? m
c,,uoA,
77 ,
-
280
I mm
n •
HRP Evans-Blue
m Figs. 2 and 3. Drawings of every third section of the right abducens nucleus in experiment 77-280 and 77-281. Black dots, EB-labelled internuclear neurons projecting to the contralateral oculomotor nucleus. Triangles, abducens motoneurons labelled with HRP. N VII, knee of facial nerve; m, midline.
examined for double-labelled neurones. However, we encountered only two distinct types of neurones, containing either H R P or EB, and no double-labelled cells were found. This is further evidence against the hypothesis that the internuclear fibers are abducens axon collaterals, especially since the efficiency of labelling in cases 77-280 and 77-281 was so high. Cell counts were performed in these two cases and the labelling of the two cell pools is reflected in the respective totals shown in Table 1. Almost the total number of abducens neurones is accounted for by the sum of the motoneurones and internuclear neurons. A small difference (not exceeding 10 %) remains and could represent a second class of interneurons projecting to another target area. Unfortunately, we were not able to obtain direct evidence because the unstained cells
185
i,,
I,
~'~
o~a
"A~
I I m
~
•
ITI 10 ~
11
.
rn
~_j~ m
~ I, 1 r a m
j
77-281 •
HRP Evons-Blue
Fig. 3. which failed to be labelled by H R P and EB markers were not identifiable, for technical reasons. The ratio of motoneurones/internuclear neurones in experiments 77-280 and 77-281 is 2:1 (Table I). The distribution of the two populations of cells was similar in all cases; both types of cells could be found throughout the whole nucleus; that is the internuclear cell pool was confined to the boundaries of the abducens nucleus, as defined by the area enclosing the motoneurons. However, in all experiments the most rostral sections contained more internuclear neurons while motoneurons predominated in the caudal parts of the nucleus (Figs. 2 and 3). This is in good agreement with physiological data 1. Computer analysis proved useful in clarifying the spatial arrangement of the cells, especially the clustering of the two types. The ratio of internuclear neurones/
186
~ ~o c
~,
~ 08
.~ 2s
0.6
~o
o
i ~ 02 o
~
o
2;0 Distance (/urn)
3'0
2;0 Distance
(/urn)
Fig. 4. Computer analysis showing the tendency of motoneurons and internuclear neurons to form separate clusters. The left side is the average value of the motoneuron/internuclear neuron ratio, plotted against the distance from a motoneuron; the right side represents the average ratio of internuclear to motoneurons plotted against the distance from an internuclear neuron. Note the decrease in the ratios with increasing distance, which reflects the formation of separate clusters. Open circles 77-280, filled circles 77-281. motoneurones in the vicinity of any given internuclear neuron drops from a relatively high value to the ratio of the two labelled cell pools within the abducens nucleus. It can be seen in Fig. 4 that within a distance of 120/am from an internuclear neuron the average ratio of internuclear neurons/motoneurons is 1.7 times higher than the overall ratio. The same is true for motoneurones. Within 120/~m from a motoneurone the ratio of motoneurones/internuclear neurones is on average 1.4 times higher than the motoneurones/internuclear neurone ratio for the whole nucleus. Thus, the tendency for the two types of cells to form separate clusters is clearly established. In spite of the successful use of Evans blue in these experiments, this dye is not a consistently sensitive retrograde tracer substance. It gave reasonably clear results when injected into the oculomotor nucleus but proved to be unsuitable for labelling the motoneurons from the lateral rectus muscle. An important advantage of EB is that axon terminals are not labelled by anterograde transport, which, in a reciprocal system such as the interconnections between abducens and oculomotor nucleus 23, might obscure the identification of double-labelled neurones by light microscopy. Preliminary experiments with [aH]HRP as second tracer 16 failed partly as a result of this. Radioactive [3H]HRP appeared to label axon terminals heavily in the abducens nucleus by anterograde transport, which obscured autoradiographic analysis. In addition there was a poor retrograde transport of the radioactivity and a relatively high enzymatic activity. The detection of weakly labelled EB neurones was highly simplified by the characteristic staining of peri- or intranuclear structures (Fig. lc and d). This phenomenon may not be present in other systems: for example vestibular neurones that were labelled in the same experiment did not show similar dark blue grains associated with the cell nucleus. In the control experiment both H R P and EB were injected into the oculomotor nucleus at a slightly different rostrocaudal localization. A small number of double-labelled neurones were found within the vestibular complex. These neurones contained distinctly blue as well as greenish brown granules.
187 The techniques used in this study have p r o v i d e d a u n i q u e o p p o r t u n i t y to establish the location, p r o p o r t i o n (2:1) a n d spatial relation o f the m o t o n e u r o n e s a n d internuclear neurones in the a b d u c e n s nucleus o f the cat. I n a d d i t i o n they p r o v i d e further evidence against the collateral hypothesis. T h e a u t h o r s gratefully a c k n o w l e d g e the e n c o u r a g e m e n t o f Prof. K. A k e r t , the valuable help o f Dr. P. Streit who developed the m e t h o d o f r e t r o g r a d e tracing with Evans blue, a n d the technical assistance b y I. G y a r m a t i , A. F~ih, D. Savini a n d R. Emch. The w o r k was s u p p o r t e d by the Swiss N a t i o n a l Science F o u n d a t i o n , g r a n t N o . 3.636.75 a n d the Dr. Eric S l a c k - G y r F o u n d a t i o n in Zfirich.
1 Baker, R. and Highstein, S. M., Physiological identification of interneurons and motoneurons in the abducens nucleus, Brain Research, 91 (1975) 292-298. 2 Bendat, J. S. and Piersol, A. G., Random Data: Analysis and Mesaurement Procedures, John Wiley, New York, 1971, pp. 28-31. 3 Bender, M.B. and Weinstein, E. A., The syndrome of the median longitudinal fasciculus. In H. W. Woltman, H. H. Merritt, S. B. Wortis and C. C. Hare (Eds.), Multiple Sclerosis and the Demyelinating Diseases, Res. PubL Ass. nerv. ment. Dis., 28 (1950) 414-420. 4 Bender, M. B. and Shanzer, S., Oculomotor pathways defined by electrical stimulation and lesions in the brainstem of monkey. In M. B. Bender (Ed.) The Oculomotor System, Harper and Row, New York, 1964, pp. 81-140. 5 Bennett, A. H. and Savill, T., A case of permanent deviation of the eyes and head, the result of a lesion limited to the sixth nucleus, with remarks on associated lateral movements of the eye balls, and rotation of head and neck, Brain, 12 (1890) 102-116. 6 Berman, A. L., The Brain Stem of the Cat. A Cytoarchitectonic Atlas with Stereotaxic Coordinates, The University of Wisconsin Press, Madison, 1968, 175 pp. 7 Blocq, P. et Guignon, G., Paralysie conjugu6e de la sixi/:me paire, Arch. Med. exp., 3 (1891) 74-89. 8 B~ittner-Ennever, J. A. and Henn, V., An autoradiographic study of the pathways from the pontine reticular formation involved in horizontal eye movements, Brain Research, 108 (1976) 155-164. 9 Carpenter, M.B., McMasters, R.E. and Hanna, G. R., Disturbances of conjugate horizontal eye movements in the monkey. I. Physiological effects and anatomical degeneration from lesions of the abducens nucleus and nerve, Arch. Neurol. (Chic.), 8 (1963) 231-247. 10 Carpenter, M. B. and McMasters, R. E., Disturbances of conjugate horizontal eye movements in the monkey. II. Physiological effects and anatomical degeneration resulting from lesions in the medial longitudinal fasciculus, Arch. Neurol. (Chic.), 8 (1963) 347-368. 11 Cogan, D. C., Kubik, C. S. and Smith, W. L., Unilateral internuclear ophthalmoplegia, Arch. Ophthal., 44 (1950) 783-796. 12 Crosby, E. C., Relations of brain centers to normal and abnormal eye movements in the horizontal plane, J. comp. Neurol., 99 (1951) 437--480. 13 Destombes, J. and Ripert, J. P., Ultrastructurai observations of the abducens nucleus of the cat after injection of horseradish peroxidase into the lateral rectus muscle, Exp. Brain Res., 28 (1977) 63-71. 14 Fuse, G., Ueber den Abduzenskern der S/iuger. In Arbeiten aus dem Hirnanatomischen lnstitut der Universitiit Ziirich, VoL V1, Bergmann, Wiesbaden, 1912, pp. 401-447. 15 Gacek, R. R., Localization of neurons supplying the extraocular eye muscles in the kitten using horseradish peroxidase, Exp. NeuroL, 44 (1974) 381--403. 16 Geisert, E. E., Jr., The use of tritiated horseradish peroxidase for defining neuronal pathways: a new application, Brain Research, 117 (1976) 130-135. 17 Gogan, P., Gueritaud, J. P., Horcholle-Bossavit, G. and Ty6-Dttmont, S., Inhibitory nystagmic interneurons. Physiological and anatomical identification within the abducens nucleus, Brain Research, 59 (1973) 410-416. 18 Graham, R. C., Jr. and Karnovsky, M. J., The early stages of absorption of injected horseradish
188
19 20
21 22
23
24 25
peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. Graybiel, A. M. and Hartwieg, E. A., Some afferent connections of the oculomotor complex in the cat: an experimental study with tracer techniques, Brain Research, 81 (1974) 543-551. Highstein, S. M., Maekawa, K., Steinacker, A. and Cohen, B., Synaptic input from the pontine reticular nuclei to abducens motoneurons and internuclear neurons in the cat, Brain Research, 112 (1976) 162-167. Konigsmark, B. W., Methods for counting neurons. In W. J. H. Nauta and S. O. E. Ebbesson (Eds.) Contemporary Research, Methods in Neuroanatomy, Springer, Berlin, 1970, pp. 315-340. Kristensson, K., Retrograde axonal flow of protein tracers. In W. M. Cowan and M. Cu6nod (Eds.), The Use t~f Axonal Transport for Studies of Neuronal Connectivity, Elsevier, Amsterdam, 1975, pp. 69-82. Maciewicz, R. J., Kaneko, C. R. S., Highstein, S. M. and Baker, R., Morphophysiological identification of interneurons in the oculomotor nucleus that project to the abducens nucleus in the cat, Brain Research, 96 (1975) 60-65. Ram6n y Cajal, S., Histologie du Systdme Nerveux de l'Homme et des Vertebras, Vol. 1 (Azoulay translation), Institute Ram6n y Cajal, Madrid, 1955, pp. 856. Warwick, R., Oculomotor Organization. In M. B. Bender (Ed.) The Oculomotor System. Harper and Row, New York, 1964, pp. 173-204.