Projection pattern of vestibulocerebellar fibers in the anterior vermis of the cat: an anterograde wheat germ agglutinin-horseradish peroxidase study

Neuroscience Letters, 74 (1987) 25 30

25

Elsevier Scientific Publishers Ireland Ltd.

NSL 04406

Projection pattern of vestibulocerebellar fibers in the anterior vermis of the cat: an anterograde wheat germ agglutinin-horseradish peroxidase study M a t s u o M a t s u s h i t a and C h a n Li W a n g * Department o['Anatomy, Institute qf Basic Medical Sciences, University o["Tsukuba, Niihari. lharaki ( Japan ) (Received I I October 1986: Accepted 14 October 1986)

Key wordsv Vestibulocerebellar projection: Cerebellar cortex: Anterior lobe; Vestibular nucleus; Cat The distribulion of mossy fiber terminals of vestibulocerebellar fibers was studied in the anterior lobe following a unilateral injection of wheat germ aglutinin horseradish peroxidase into the medial and descending vestibular nuclei in the cat. In the transverse plane 3 groups of labeled terminals were seen bilaterally: group I located in the midline region, group 2 at 0.5 mm, and group 3 between 0.75 and 1.5 mm lateral to the midline. Reconstruction of the distribution in the horizontal plane revealed that the labeled terminals were distributed in 3 areas extending apicobasally in the basal part of the lobules.

Studies using the retrograde horseradish peroxidase method have established that secondary vestibulocerebellar fibers terminate not only in the nodulus, uvula and flocculus, but more widely in the anterior lobe and lobules VI VIII of the posterior lobe [2, 3, 5 7, 11, 13, 17]. These projections arise from the superior (SVN), medial (MVN) and descending vestibular nuclei (DVN). Projections from the lateral vestibular nucleus described previously [11] were not recognized [6, 7, 13]. A wide mediolateral distribution of vestibulocerebellar fibers has been described, which extends as thr as the lateral part of lobules V and VI [11], crus I and II, and the paramedian Iobule [6]. Following injections of wheat germ agglutinin horseradish peroxidase (WGA HRP) into the vestibular nuclei in the rabbit, Epema et al. demonstrated that vestibulocerebellar fibers terminate in several longitudinal zones in lobule IX, but diffusely in Iobule X [4]. However, no previous studies using the anterograde tracing technique have determined the terminal field of secondary vestibulocerebellar fibers in each lobule of the cerebellar cortex. Following injections of WGA HRP into the DVN and the MVN, we examined the distribution of labeled secondary vestibulocer*Present address: Department of Anatomy, China Medical College, Shien Yang, People's Republic of China. Correspondence: M. Matsushita, Institute of Basic Medical Sciences, University of Tsukuba, Niihari, lbaraki 305, Japan.

0304-3940,,'87,,'$ 03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd.

26

ebellar fibers in the anterior lobe and lobule VI of the posterior lobe. In this study. the projection field in the horizontal plane was reconstructed, based on the distribution in transverse or cross-sections through each lobule. The results of experiments in two cats, which had been used in a previous study [15], were selected for description. A total of 0.07 #1 (case no. 787) or 0.1 /1t (case no. 717) of 2% WGA conjugated to H R P (Sigma or Toyobo) was injected through a glass micropipette into the descending or/and medial vestibular nuclei. After 3 days the animals were killed under deep pentobarbital anesthesia by perfusion of 2.5% glutaraldehyde and 0.5% paraformaldehyde in phosphate buffer. Cerebella and brainstem were removed and stored in a 10% sucrose-buffer mixture. Brainstems were cut transversely and sections through the injection sites were reacted with diaminobenzidine according to the method of Adams [1]. One cerebellum (no. 717) was cut serially at 100 Ftm into transverse sections while in the other each lobule of the anterior lobe and lobule VI were detached from the corpus medullare and cut serially at 100/~m into cross-sections in the apicobasal direction. These sections were mounted on gelatin-chrome-alum-coated slides and reacted with tetramethyl benzidine according to the method of Mesulam et al. [16]. Distribution of labeled mossy fiber terminals in each section was plotted serially on a sheet of section paper and reconstructed in the horizontal plane. Details of histological procedures have been described previously [15, 20]. The extent of injections in cases nos. 717 and 787 has been described in detail [15]. The injected W G A - H R P in case no. 717 spread over the medial part of the DVN, and the entire part of the MVN and the prepositus hypoglossi nucleus at levels P 10.0-10.8 (Fig. IA); it also diffused into the adjacent reticular formation. At level P 9.2 only the MVN was stained with the W G A - H R P . In case no. 787 the W G A -

A

B

(7 1 7)

(787)

D

M Ph

Fig. I. Photomicrograph and diagram showing the extent of W G A - H R P injections into (A) the medial part of the descending vestibular nucleus and the entire part of the medial vestibular nucleus (no. 717), and (B) the descending vestibular nucleus (no. 787), IO, inferior olivary nucleus; D and M, descending and medial vestibular nucleus; Ph, nucleus prepositus hypoglossi; Vi, subnucleus interpolaris of the spinal nucleus of the trigeminal nerve.

27 B

A

(4.4 mm) I

Ilia-Ill

RostraL

_[

t

"
IIIb-III",,.,,,,,,.,J ~ ~ Ilia-Ill mm

rs

,

6

IIIb Ilia

,

5

_'

"-;-: -'-"~

. . . . . . . . . "-::L:

I

Caudal

IIIb-III

4

,

5

, ,j 2

Apical o

1 I

0

--1

--2

1 _

•

2

3..-i :" ."::]:.." .~"'::

J

~"

.....:.Ji;;:.:":.: ;/::-.,. "..." , ": .'. "".5 ..;'::..

u

"'"

-.:. - ...11.'.: :.::

S,'.."

p

5

"4

6

Basal Fig. 2. Diagram showing the distribution of labeled mossy fiber terminals (dots) in the left half of subtobule llIb (case no. 717). A: left parasagittal and caudal view of lobule III to show the plane of section in B. The side facing lobule 1V is designated as the caudal side (cs), and that facing lobule II as the rostral side (rs). B: distribution of labeled mossy fiber terminals (dots) in a transverse section at 4.4 m m from the apex (no. 717). C: reconstruction of the distribution on the caudal side of sublobule IIIb to lobule 11I. N u m b e r s 1 3 in the transverse plane (B) are groups of labeled terminals and those in the horizontal plane (C) are areas of projection. Arrowheads indicate the plane of section in B. Horizontal and vertical scales (mm) indicate the mediolateral distance from the midline of the lobule (vertical line through zero) and the apicobasal distance (zero at the apex of the lobule), respectively. Line marked by the asterisk denotes the apex of a small folium protruding in the middle of the lobule.

28 H R P was deposited in the dorsal two-thirds of the DVN and the most medial part of the MVN (Fig. IB). Distribution of labeled terminals was examined in all lobules from lobules I to VI in these cases, but the findings made in sublobules Ill and IV of case no. 717 will be described to show the typical distribution pattern of labeled terminals. The labeled mossy fiber terminals were distributed bilaterally in the cerebellar cortex after a unilateral injection. Clusters of labeled terminals seen in the transverse plane were termed 'groups' and numbered• Correspondingly, projection fields could be reconstructed in the horizontal plane of the lobule by tracing these groups in a series of transverse sections. These projection fields were termed 'areas' and also numbered, as had been done in a previous study [20]. In the transverse plane the labeled terminals appeared mainly in sections through middle to basal parts of the lobules. Few labeled terminals were seen in sections through apical parts• In a section through lobule III at 4.4 mm from the apex (Fig. 2A, B), 3 groups of labeled terminals were seen: (I) group I located in the midline region, (2) group 2 located at

mm 6

I

5

IVb-lV

I

(caudal

4

3 ,

Apical o

1

2

I

i

'

l

I

side)

o

--I

I

.°

/ f,

•

2

":'.

•

°.

','.

::i

--2

:." "" "'- '""

--3

,:.,.

:.:..:'...

'.'

:

.'.;::.: • ' ::.,-'." )

. .

:':."

,

,

,

I

,

I

-:..

":

z." "- :::"• .:°'"

---:. :~!4.1.~ ;. ."

,

l

:...

,

.. " "-....--4-

:

I-6

Basal

Fig. 3. Diagram showingthe distribution of labeled terminals on the caudal side of sublobule IVb to lobule IV, reconstructed from a series of transverse sections.

2~

around 0.5 mm lateral to the midline, and (3) group 3 located between 0.75 and 1.75 mm from the midline. Reconstruction of the distribution on the caudal side of sublobule I I I b to lobule III revealed that these 3 groups of labeled terminals formed longitudinal projection areas in the horizontal plane of this lobule (Fig. 2C): (l) area 1 extending in the apicobasal direction along the midline, (2) area 2 extending between 0.25 and 0.5 ram, and (3) area 3 extending mainly between 0.75 and 1.25 mm lateral to the midline. These 3 areas extended for about 3 m m from the basis in the apicobasal direction. On the caudal side of sublobule IVb to lobule IV, 3 projection areas were also recognized (Fig. 3). Although they were broader and less demarcated, they appeared to correspond, respectively, to the 3 areas observed in Iobule IIl (Fig. 2C). As a whole, these areas were confined to the basal part of the vermis, area I extended for about 4 mm in the apicobasal direction. Areas 2 and 3 extended less than 3.0 mm from the basis of the lobule. As the number of labeled terminals increased in caudal Iobules (lobules V to VI), the boundaries of the areas became less distinct. Nevertheless, the projection pattern appeared common to all the lobules examined. The present study reveals the projection field of the DVN (no. 787) or:and the MVN including the prepositus hypoglossi nucleus (no. 717). The projection field of case no. 717 may include that of the nucleus preposilus hypoglossi which also pro.jeers to the anterior lobe [2, 6, 10, 13]. However, the findings were similar in these two cases although the projection pattern observed in case no. 787 was less conspicuous because of a limited injection into a single nucleus. The significant finding in the present study is that the MVN and the D V N project to 3 longitudinal areas: (I) area I extending in the midline region (zone At), (2) area 2 located from zones At to A> and (3) area 3 in zone A2 to the medial part of zone B of Voogd [18]. They were confined to the basal part of the lobules. This projection pattern (Figs. 2 and 3) resembled that of the spinocerebellar tract arising from the central cervical nucleus [20]. A close relationship between the vestibular nuclei and the central cervical nucleus has been demonstrated anatomically and electrophysiologically. Lobules 1 and I I of the anterior lobe, which are the main projection sites of the central cervical nucleus [8, 12, 14, 19], receive input from secondary vestibulocerebellar fibers [7, II, 13]. Furthermore, a certain population of neurons of the central cervical nucleus are excited by stimulation of the contralateral anterior or posterior semicircular canals and inhibited by stimulation of the ipsilateral counterparts in the opposite plane [9]. These findings suggest that the input from the vestibular nuclei and the central cervical nucleus may be integrated in the specific longitudinal areas of the vermis of the anterior lobe and lobule VI of the posterior lobe. Wc thank H. Matsuura for his unfailing technical assistance and M. Teranishi for his photographic assistance. This study was supported by Grant 60304041 from the Ministry of Education. Science and Culture of Japan. 1 Adams. J.C., f leavy metal intensification of l)AB-based H R P reaction product. J. Histochem. ('_,,toctlem., 29 ( 1981 ) 775. 2 Batini, ('., Buisseret-Dehnas, C., Corvisier, ,I,. Hardy, O. and Jassik-Gerschcnfcld, D., Brain slem nuclei g i v i n g l i b e r s t o l o b u l e s V l a n d V l I o f t h e c e r e b e l l a r v e r m i s , Brain Res.,153 (1978) 241 261.

30 3 Carleton, S.C. and Carpenter, M.B., Afferent and efferent connections of the medial, inferior and lateral vestibular nuclei in the cat and monkey, Brain Res., 278 (1983) 2%51. 4 Epema, A.H., Guldemond, J.M. and Voogd, J., Reciprocal connections between the caudal vermis and the vestibular nuclei in the rabbit, Neurosci. Lett., 57 (1985) 273-278. 5 Frankfurter, A., Weber, J.T. and ttarting, J.K., Brainstem projections to lobule VII of the posterior vermis in the squirrel monkey, as demonstrated by the retrograde axonal transport of tritiated horseradish peroxidase, Brain Res., 124 (1977) 135-139. 6 Gould, B.B., Organization of afferents from the brain stem nuclei to the cerebellar cortex in the cat, Adv. Anat. Embryol. Cell Biol., 62 (1980) 1 79. 7 Hirai, N., Vestibular afferent inputs to lobules I and II of the cerebellar anterior lobe vermis in the cat, Brain Res., 277 (1983) 145 149. 8 Hirai, N., Hongo, T. and Sasaki, S., A physiological study of identification, axonal course and cerebellar projection of spinocerebellar tract cells in the central cervical nucleus of the cat, Exp. Brain Res., 55 (1984) 272 285. 9 Hirai, N., Hongo, T., Sasaki, S. and Yoshida, K., The neck and labyrinthine influences on cervical spinocerebellar tract neurones of the central cervical nucleus in the cat, Prog. Brain Res., 50 (1979) 529 536. 10 Kotchabhakdi, N., Hoddevik, G.H. and Walberg, F., Cerebellar afferent projections from the perihypoglossal nuclei: an experimental study with method of retrograde axonal transport of horseradish peroxidase, Exp. Brain Res., 31 (1978) 13 29. 11 Kotchabhakdi, N. and Walberg, F., Cerebellar afferent projections from the vestibular nuclei in the cat: an experimental study with the method of retrograde axonal transport of horseradish peroxidase, Exp. Brain Res., 31 (1978) 591 604. 12 Matsushita, M. and Okado, N., Spinocerebellar projections to lobules I and II of the anterior lobe in the cat, as studied by retrograde transport of horseradish peroxidase, J. Comp. Neurol., 197 (1981) 41 l~424. 13 Matsushita, M. and Okado, N., Cells of origin of brainstem afferents to lobules 1 and II of the cerebetlar anterior lobe in the cat, Neuroscience, 6 (1981) 2393 2405. 14 Matsushita, M., Tanami, T. and Yaginuma, H., Differential distribution of spinocerebellar fiber terminals within the lobules of the cerebellar anterior lobe in the cat: an anterograde WGA HRP study, Brain Res., 305 (1984) 157 161. 15 Matsushita, M. and Wang, C.L., Cerebellar corticovestibular projections from lobule IX to the descending vestibular nucleus in the cat. A retrograde wheat germ agglutinin-horseradish peroxidase study, Neurosci. Lett., 66 (1986) 293 298. 16 Mesutam, M.-M., Hegarty, E., Barbas, H., Carson, K.A., Gower, E.C., Knapp, A.G., Moss, M.B. and Mufson, E.J., Additional factors influencing sensitivity in the tetramethyl benzidine method for horseradish peroxidase neurohistochemistry, J. Histochem. Cytochem., 28 (1980) t 255-I 259. 17 Precht, W., Volkind, R. and Blanks, R.H.I,, Functional organization of the vestibular input to the anterior and posterior cerebellar vermis of cat, Exp, Brain Res., 27 (1977) 143-160. 18 Voogd, J., The importance of fiber connections in the comparative anatomy of the mammalian cerebellum. In R. Llinas (Ed.), Neurobiology of Cerebellar Evolution and Development, American Medical Association Education and Research Foundation, Chicago, 1969, pp. 493-514. 19 Wiksten, B., The central cervical nucleus in the cat. IIl. The cerebellar connections studied with anterograde transport of 3H-leucine, Exp. Brain Res., 36 (1979) 175 189. 20 Yaginuma, H. and Matsushita, M., Spinocerebellar projection fields in the horizontal plane oflobules of the cerebellar anterior lobe in the cat: an anterograde wheat germ agglutinin-horseradish peroxidase study, Brain Res., 365 (1986) 345 349.