A Study of Afferent Input to the Inferior Olivary Complex in the Rat by Retrograde Axonal Transport of Horseradish Peroxidase JAMES T. BROWN,' VICTORIA CHAN-PALAY AND SANFORD L. PALAY Departments of Anatomy and Neurobcology, Haruard Medical School, Boston, Massachusetts 021 15

ABSTRACT Under direct visualization, minute amounts of horseradish peroxidase were injected by controlled air pressure through glass micropipettes into the inferior olive of t h e albino rat. Well localized unilateral injections were obtained, without damage to other brainstem areas or spread into the adjacent reticular formation. After 24 to 36 hours, t h e entire brain and spinal cord were examined by light microscopy for labelled neurons supplying afferents to the inferior olive. With few exceptions, labelled somata rostra1 to the inferior olive were found ipsilateral to the injection site, t h e greatest number occurring in t h e subparafascicular nucleus and central gray substance around t h e caudal third ventricle, in t h e N. of Forel's field, Nn. of Darkschewitsch and Cajal, and adjacent reticular formation, and in t h e Edinger-Westphal nucleus. Labelled cells were also noted in t h e sensorimotor cortex, red nucleus, peri-aqueductal gray and all subdivisions of the pretectal complex. Caudal to the olive, labelled neurons were located primarily on t h e contralateral side in the lateral reticular nucleus, dorsal column nuclei and nucleus proprius of the spinal cord. At t h e level of the olive, numerous labelled cells occurred bilaterally, but mainly contralateral to the injection site in t h e lateral and interpositus nuclei of t h e cerebellum. A smaller number were seen in t h e N. prepositus hypoglossi and in the medial and spinal subdivisions of t h e vestibular complex ipsilaterally, in the N. spinal tract of V contralaterally, and in the gigantocellular reticular nucleus bilaterally. A few neurons in t h e N. raphe obscurus were also labelled. No positive cells were found in t h e caudate nucleus, globus pallidus, locus coeruleus, or medial cerebellar nucleus. The results are compared and contrasted, region by region, with previous studies of afferent olivary pathways and, where possible, correlations are made with physiologic data. With t h e exception of collateral branches to Deiters nucleus (It0 et al., '66; Allen et al., '72a,b; Groenewegen and Voogd, '761, inferior olivary neurons project their axons in orderly fashion primarily to t h e contralateral cerebellar cortex (Brodal, '40, '76; Armstrong et al., '74; Brodal e t al., '75; Courville, '75; Hoddevik e t al., '76; Groenewegen and Voogd, '76) and, en route, furnish collaterals to t h e deep cerebellar nuclei (Eccles e t al., '67; Ito e t al., '70; Matsushita and Ikeda, '70; Eller and ChanPalay, '76; Groenewegen and Voogd, '76). In all likelihood, the inferior olive represents t h e J. COMP. NEUR., 176: 1-22.

major source of climbing fibers to t h e cerebellum (Szentagothai and Rajkovits, '59; Eccles e t al., '66; Desclin, '74). The functional significance of this olivo-cerebellar pathway has been t h e subject of numerous physiologic I Supported in part by Research Grants NS10536 and NS03659 and Training Grant NS05591 from the National Institute of Neurological and Communicative Disorders and Stroke James T. Brown LS a postdoctoral trainee supported by Training Grant NS05591 from the National Institute of Neurological and Communicative Disorders and Stroke. Present address. Division of Neurological Surgery, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago. Illinois 60611 Please send reprint requests to Victoria Chan-Palay, Department of Neurobiology, Harvard Medical Schoal, 25 Shattuck Street, Boston, Massachusetts 02115.

1

2

J. T. BROWN, V. CHAN-PALAY AND S. L. PALAY

studies over the past ten years, and during this time many theories have emerged. These have been amply reviewed by Armstrong ('74) and Chan-Palay ('77) and will not be further recounted here. What has been lacking, however, is a more precise determination of the various sources of afferents to the inferior olive by which this very important pathway might be influenced. Early anterograde degeneration studies with Marchi and reduced silver techniques began the piecemeal assembly of olivary afferents, but even discrete lesions ran the risk of destroying fibers of passage as well as cell bodies, and there was often controversy over exactly what constituted terminal degeneration. Autoradiography has overcome most of these difficulties, but the problem of limitation of injection sites remains, and this method still enables determination of only one afferent source a t a time, although i t is extremely useful in defining precisely which regions of a nucleus like the olive receive afferents from a particular source. The method of retrograde axonal transport of horseradish peroxidase (HRP) as developed by Kristensson and Olsson (Kristensson and Olsson, '71, '73a,b; Kristensson, '75) and by the LaVails (LaVail and LaVail, '72, '74; LaVail, '75a,b) has provided an ideal means for more definitive localization of olivary afferent sources. Within the past year, HRP injections into the inferior olive have enhanced our knowledge of the cerebellar nuCleo-olivary pathway (Martin et al., '76b; Tolbert e t al., '761, and attempts have also been made to delineate the meso-diencephalic sources of olivary afferents (Henkel et al., '75; Linauts and Martin, '76). With the exception of the study by Bishop et al. ('76a) in the cat, this technique has not been used to define all possible sources of afferents to the olive. Also, the need for precise injections that are confined to the olive is readily apparent. To this end, we employed a ventral para-pharyngeal approach to the inferior olive of the rat, thereby avoiding damage to the overlying cerebellum and dorsal medulla. When injections limited to the boundaries of the olive were accomplished, the entire brain and spinal cord were searched for labelled neurons. MATERIALS AND METHODS

Eighteen female Sprague-Dawley rats (200250 gm) were used for unilateral olivary HRP injections, of which seven were found suit-

able for studying olivary afferents (animals K7637, K7638, K7639, K7642, K7643, K7646, K7648). One rat (K76471, in which the entire surgical and histological procedure was carried out, but without an injection of HRP into the brain, served as a control. The animals were anesthetized with a n intraperitoneal injection of 35% chloral hydrate (0.1 m1/100 gm body weight). They were then placed supine in a Horsley-Clarke stereotaxic apparatus with the long axis of the head a t about 35" from horizontal. A mixture of 95% O 2 and 5% CO, was administered at 3 l/min throughout the procedure. The animals were wrapped in a heating pad to prevent hypothermia and, intermittently, 5 ml of normal saline were given subcutaneously to maintain hydration. A 5 cm vertical paramedian incision was made, extending from the angle of the mouth to the clavicle. The thyroid was dissected and reflected medially. The strap muscles were then excised and the trachea and carotid sheath carefully freed. The longus capitus muscle was partially excised, thus exposing the anterior arch of C-1 and the basiocciput from the midline laterally to the hypoglossal foramen. With the trachea under direct visualization, endotracheal intubation through the mouth was accomplished with a firm 6 cm polyethylene tube (Intramedic #PE200, O.D. 1.9 mm), which was left in place for the remainder of the surgical procedure. Next, while retracting the trachea medially, two burr holes were made in the basi-occiput with a dental drill, and the craniectomy was enlarged with forceps and small hemostat. The anterior arch of C-1 was left intact. After opening the dura and arachnoid, the basilar artery, pyramid, and hypoglossal rootlets were easily visualized. In general, the hypoglossal rootlets outlined the rostro-caudal extent of the olive. Injections into the olive could be made either through or just lateral to the pyramid, depending upon where in the medial to lateral extent of the olive the injection site was desired. All injections were made with glass micropipettes (Kimax-51" #46485, O.D. 0.7-1.0 mm) drawn on a Kopf vertical pipette puller (model 700C). The pipettes, with tips 10-15 p m in diameter, were filled by capillary action with a 7%solution of HRP (either Sigma type VI or Worthington HPOFF 56D530) in normal saline and attached to a n air-pressure injection apparatus mounted on a stereotaxic probe drive. The trachea was retracted

AFFERENTS TO RAT INFERIOR OLIVE, HRP

3

silateral olive without spread dorsally into the adjacent reticular formation (fig. 1). Although no one injection encompassed the entire olive, the series as a whole nearly did so. The exceptions were the most medial aspect of the medial accessory olive throughout its rostro-caudal extent and the rostral tip of the dorsal accessory olive. Two additional experiments (K7638, K76391, in which the HRP spread beyond the confines of the ipsilateral inferior olive, were used only to confirm the locations of positively labelled cells found in the five successful studies. Since endogenous peroxidase activity in the rat brain has been reported by Sherlock and Raisman ('75) and more recently by Keefer and Christ ('761, another experiment (K7647) was performed in which the entire surgical and histological procedure was carried out but no peroxidase was injected into the brain. In this control animal, no labelled neurons were found in areas containing HRP-positive neurons in the other experiments. Therefore, it is concluded that the sources of afferents reported here are labelled because of retrograde axonal transport of HRP from the olive and not because of endogenous peroxidase within the neuronal somata. The results obtained in these experiments are in agreement with previous anatomic and physiologic studies which have, over the years, pieced together the various sources of olivary afferents. In addition, however, the present work yields some new and rather interesting qualitative as well as quantitative data. A striking feature of the results (table 1) is that the sources of olivary afferents rost r a l to the olive a r e almost exclusively ipsilateral to the injection site, whereas those caudal to the olive are contralateral. Afferents a t the approximate level of the olive tend to be bilateral, but predominantly contralateral, in origin. K7643 The most extensive yet localized injection was in animal K7643, in which the entire principal olive (PO) and most of the dorsal accessory olive (DAO) were heavily stained with HRP. The medial and the rostral tips of the DAO and the majority of the medial accessory olive (MA01 were unstained, howRESULTS ever (fig. 1). In this experiment the cerebral cortex ipsiFive experiments (K7637, K7642, K7643, K7646, K7648) were judged successful in that lateral to the injected olive contained many the HRP injection was confined to the ip- labelled cells, while on the contralateral side

medially, and the entire injection apparatus was lowered onto the surface of the medulla. At a depth of about 0.4 mm from the surface, 3-10 psi of air pressure was gradually applied, and 0.05-0.10 p1 of HRP was delivered into the olive over a period of three to five minutes. The pipette was then immediately withdrawn from the brain to avoid further trauma and edema. The endotracheal tube was removed and the skin was closed with a running suture of 5-0 nylon. Postoperatively, the animals were warmed under a heat lamp and subcutaneous injections of normal saline were given. After a survival period of twenty-four to thirty-six hours, the animals were perfused transcardially with a mixture of 1%formaldehyde and 1%glutaraldehyde in 0.12 M phosphate buffer (pH 7.4, 22°C) (Palay and ChanPalay, '74). The brain and entire spinal cord were immediately dissected and left overnight in fixative a t 4"C, after which they were transferred to a solution of 0.05 M phosphate buffer and 30% sucrose (pH 7.4, 4°C) for 48 hours. The tissue was then cut transversely at 30 p m on a freezing microtome into a solution of 0.05 M tris-HC1 buffer and 5% sucrose (pH 7.6, 22°C). The histochemical reaction for demonstrating peroxidase was essentially that of Graham and Karnovsky ('66). Every third section (every section through the injection site) was mounted from a 0.5% gelatinalcohol solution. The sections were then dehydrated, defatted, and lightly counterstained with 0.1%cresyl violet. Each mounted section through the brain and spinal cord was then carefully scanned a t 200 X magnification under brightfield illumination. All labelled neurons were counted and examined for location, laterality, relative size and HRP uptake. The atlas of Sidman et al. ('71) was used for reference. Serial sections through the injection sites were drawn with a projection apparatus and the distribution of HRP at the injection site and any fibers indicative of diffuse labeling were plotted under the microscope and entered on the drawings. To facilitate comparisons of the separate cases, the results of each case were transferred to standardized diagrams of the inferior olivary complex in coronal sections.

4

J T. BROWN, V. CHAN-PALAY AND S. L. PALAY Abbreviations

BC, Brachium conjunctivum CA. Cutaneous afferents CC, Cerebellar cortex DCN. Dorsal column nuclei DN. Deep nuclei

DRG, Dorsal root ganglion FRA, Flexor reflex afferents GRN, Gigantocellular reticular nucleus 10, Inferior olive LCN, Lateral cerebellar nucleus

LRN, Lateral reticular nucleus NP, Nucleus proprius PTC, Pretectal complex RB, Restiform body SC, Spinal cord

caudal

K7643

K7648

K7637

K7642

K7646

A,&&&

I dorsal accessory olive

rostra1

0 medial accessory olive prrnciDal olive

Ez3

Fig. 1 Precise locations of injection sites in five experimental cases (K7643, K7648, K7637, K7642, K7646) plotted onto reference coronal sections through t h e inferior olivary complex of the rat. Note t h a t t h e injections ar e all small but vary in their placements within the various subdivisions of t h e olivary nucleus. Only in one case, K7648. was diffuse labelling of a number of axons seen in t h e ipsilateral pyramid.

only a n occasional cell was seen (table 2). The positive cells, which were confined to layer V of the sensorimotor cortex, were quite large and contained numerous HRP granules. One of the greatest sources of olivary afferents

was around the caudal third ventricle where the ipsilateral subparafascicular nucleus and central gray substance contained numerous small to medium sized cells of moderate HRP content. This high concentration of exclusive-

AFFERENTS TO RAT INFERIOR OLIVE. H R P

P

s a .-

5

ly ipsilateral labelled cells continued caudally into the N. of Forel’s field, the N. of Darkschewitsch, the interstitial N. of Cajal, the adjacent rnesencephalic reticular formation, and the Edinger-Westphal nucleus. All cells in this region were small to medium sized, with slight to moderate HRP content. In this experiment, these peri-ventricular and periaqueductal neurons accounted for almost 43%) of all labelled cells counted. The pretectal complex contained a large number of labelled neurons, again, all ipsilatera1 to the injection site. The cells found here were large with heavy HRP labelling, and most were located in the caudolateral part of the anterior pretectal nucleus. Surprisingly, t h e red nucleus contained only a few small t o medium sized positive cells with a relatively light HRP content. They were situated in the dorsomedial part of the nucleus and all were ipsilateral to the injected olive. A few small, lightly labelled ipsilateral neurons were also found in the dorsolateral peri-aqueductal gray. As noted in table 2, no labelled neurons were found in the caudate or pallidum on either side. The lateral reticular nucleus contralateral to the injection site consistently contained a few cells of medium to large size with modera t e HRP labelling. More labelled cells were seen in the dorsal column nuclei, particularly in t h e medial cuneate nucleus. These cells were of variable size and HRP content, and all were located contralateral to the injected olive, save one gracile nucleus neuron which occurred on the ipsilateral side. Many positive neurons were found in the spinal cord in the medial aspect of t h e nucleus proprius. These cells were of variable size but all were rather heavily labelled with HRP, and all were noted contralateral to the injection site. Although numerous sections throughout the length of t h e spinal cord were examined, no labelled neurons were found below C-4. In addition to afferents originating around the third ventricle and aqueduct, the deep cerebellar nuclei were found to be the other principal source of olivary afferents. Both the lateral and the interpositus nuclei contained a great number of labelled cells; the lateral nucleus, however, contained about twice as many positive neurons as t h e interpositus. Although these neurons were found bilaterally in each nucleus, t h e vast majority were “ T h e nomenclature applied to this particular group uf nnclri has been quite varied i n previous reports The terminology uspd here will he t h a t of Scalia 1’721

6

J T BROWN. V CHANPALAY AND S L PALAY TABLE L

Locations and incidence of labelled neurons following uniluterai H R P injection into rat inferior o l i ~ (K7643l e Nucleus

1. Cerebral cortex 2. Subparafascicular nucleus 3. Peri-(thirdlventricular gray 4. Zona incerta 5. N. of Forel's field 6 Nn. of Darkschewitsch. Cajal. a n d adjacent reticular formation 7. Edinger-Westphal nucleus 8 N. of optic tract 9. Posterior pretectal nucleus 10 Olivary pretectal nucleus 11. Anterior pretectal nucleus 12. Red nucleus 13. Peri-aqueductal gray 14. Gigantocellular reticular nucleus 15. Lateral cerebellar nucleus 16 lnterpositus nuclei of cerebellum 17. N. raphe obscurus 18 Vestibular nuclei (medial & spinal1 19. Prepasitus nucleus of hypoglossal 20. N. spinal tract of V (interpolar) 21. Lateral reticular nucleus 2 2 Medial cuneate nucleus 23. Gracile nucleus 24 N. proprius (spinal c o d

Ip*ilateral

Midlinr

'

('ontralaternl

2+ 3+ 412+ 34+ I t 1+ 1+ 2+ 1+ 1+

l+ It I+

I+ 8+

4T l+

1+ 1+

1+

1+ 1+ 2+ l+

2t

P e r t i n e n t nredtive findings

I . Caudate nucleus 2. Globus pallidus 3. Locus coeruleus 4. Medial cerebellar nucleus Plus marks Indicate t h e iricidpnce of labelled neurons in e a c h n u c l e u b relative t o t h e contralateral lateral cerebellar nucleus u h r h contained t h e greatest n u m b e r 18471 of labelled neurons counted In t h l s experiment P e r t i n e n t negatives include those areas not labelled but which have been reported In earlier s t u d i e s to project to t h e o l i \ r ' Keglons in which only one ur two cells were found In t h i s rxperrment include t h e N cuneiformis. superior colliculus ldeep laycri. v e n t r a l nucleus of t h e lateral lemmscus. mesencephalic nucleus of V a n d N . centralis c a u d a h s pontis i p s i l a t e r a l l ~ and , t h e facial nucleus.. N of t h e s o l i t a n , tract. N centralis medullae ohlongatae ( v e n t r a l p a r t i . a n d dorsal m a t o r nucleus of X contra laterall?

located contralateral to t h e injected olive. Within t h e lateral nucleus, most labelled neurons were small, ranging from 8-12 p m i n width and 10-15 p m in length, and had angular or fusiform somata. They contained few HRP granules, and were primarily situated in the medial hilus zone of the nucleus. Other positive cells were slightly larger with angular, more heavily labelled somata, and were found in the dorsal and caudolateral portions of the lateral nucleus. Labelled cells within the interpositus were also rather small and angular but varied more in HRP content. Although scattered throughout the nuclei, they were mainly located in t h e lateral aspect of both the anterior and t h e posterior interpositus. In experiment K7643, almost 42% of all labelled neurons counted were found in t h e deep cerebellar nuclei, 28.2%situated in the

contralateral lateral nucleus, 0.9% in the ipsilateral lateral nucleus, 12.4%in the contralateral interpositus, and 0.2% in the ipsilateral interpositus. No labelled cells were found in t h e medial cerebellar nucleus on either side, however, and no Purkinje cells contained HRP. The medullary reticular formation contained a few large, moderately labelled neurons bilaterally in the gigantocellular nucleus, but most of these were contralateral to t h e injection site. Within t h e interpolar region of the N. spinal tract of v, there were several medium to large, rather heavily labelled cells contralateral to t h e injected olive. Both t h e medial and the spinal subdivisions of t h e ipsilateral vestibular complex contained a few large neurons with heavy HRP labelling. In the prepositus nucleus of t h e hypoglossal,

7

AFFERENTS TO RAT INFERIOR OLIVE, HRP TABLE :3

Locations and incidence of labelled neurons followmgunilateral HRPinjection into rat infenor olive (K7648) ' Nucleus

lpsilateral

1. Cerebral cortex 2 . Subparafascicular nucleus 3. Peri-(third)ventricular gray 4. N. of Forel's field 5. Nn. of Darkschewitsch,Cajal, and adjacent reticular formation 6. Edinger-Westphalnucleus I. Red nucleus 8 . Lateral cerebellar nucleus 9. Inferior olive 10. Medial cuneate nucleus 11. Gracile nucleus

1+ 2+ 2+ 1+

Midline

Contralateral

2+

2+ 1+

If If

If l+

P e r t i n e n t negative findings-as in tahle 2 Plus m a r k s indicate t h e incidence of labelled neurons in each nucleus relative to t h e ipsilateral N n . of Darkschewltsch, CaJal, a n d adjacent reticular formation which contained t h e g r e a t e s t n u m h e r I361 of labelled neurons counted in t h i s experiment. ' Regions in which only o n e labelled cell w a s found include t h e peri-aqueductal g r a y ipsilaterally. a n d t h e a n t e n o r interpositus nucleus of t h e cerebellum, dorsal motor nucleus of X a n d l a t e r a l reticular nucleus contralaterally In t h i s experiment. several neurons with diffuse labellmg were found in t h e contralateral medial accessory olive Srr text.

also ipsilateral to the injection site, there were some small cells w i t h a moderate amount of HRP. Finally, the raphe obscurus contained a few small, heavily labelled neurons; however, labelled neurons were found in no other raphe nuclei. The locus coeruleus was not labelled. K7648 In this experiment, t h e entire PO was stained with HRP, a s was t h e mid region of the DAO and lateral aspect of t h e M A 0 throughout most of their rostro-caudal extent (fig. 1). The total number of labelled neurons was much smaller in this experiment t h a n in K7643, however, and the labelled cells were confined to fewer nuclei (table 3). Layer V of the ipsilateral sensorimotor cortex contained several large cells labelled with numerous HRP granules. As in K7643, however, most of the labelled neurons occurred in t h e ipsilateral subparafascicular nucleus and central gray around t h e caudal third ventricle and, more caudally, in the Nn. of Darkschewitsch, Cajal and adjacent reticular formation and in the Edinger-Westphal nucleus. The N. of Forel's field and t h e red nucleus ipsilateral to the injected olive also contained a few labelled cells. All of these positive cells were small to medium sized and contained a slight to moderate amount of label. The pretectal complex was not labelled. Contralateral to t h e injection site, the medial cuneate and gracile nuclei contained a few small cells with light HRP labelling. No

positive cells were found in the lateral reticular nucleus or nucleus proprius of the spinal cord in this experiment, however. The medial hilus zone of the lateral cerebellar nucleus contralateral to t h e injected olive contained several small cells with light peroxidase labelling, but none was found in the ipsilateral lateral nucleus or in the interpositus nuclei on either side. The total number of labelled neurons in the contralateral lateral cerebellar nucleus was markedly less in K7648 than in K7643. In all experiments, t h e contralateral olive was devoid of typically labelled cells, although in this experiment, a few neurons in the contralateral medial accessory olive were found diffusely stained with HRP. In light of evidence presented by Nauta e t al. ('741, LaVail and LaVail ('741, and LaVail ('75a,b), this staining was interpreted a s secondary to t h e injury of contralateral olivary axons passing through the injection site. Such diffusely labelled cells were not found i n t h e cerebral cortex. However, i t is thought by some authors (Kristensson and Olsson, '74; DeVito et al., '74; LaVail, '75a,b; Halperin and LaVail, '75) t h a t neuronal cell bodies whose axons pass through a n injection site and are subsequently injured by t h e injection can become typically labelled with HRP. This may be significant with regard to some or all of t h e labelled neurons found in t h e cerebral cortex in this experiment, since t h e pyramid was heavily stained with HRP. As indicated in figure 1, however, t h e pyramid was not stained in the

8

J

T BROWN. V CHANPALAY AND S L PALAY TABLE 4

Locations and incidence of labelled neurons following unilateral HRPin~ectioninto rat inferior olive lK7637 I Nucleus

1. Cerebral cortex 2. Subparafascicular nucleus 3. Peri-(third1ventricular gray 4. Nn. of Darkschewitsch, Cajal. and adjacent reticular formation 5. Edinger-Westphal nucleus 6. Lateral cerebellar nucleus

lpsilateial

Midlint.

C'ontralnteral

1-

4-

2+

2+ 2+

2+ l i

P e r t i n e n t negative findings-as i n table 2 Plus marks indicate t h e incidence of labelled neurons in each nucleus relative to t h e Ipailateral cerehral w r t w which contacnrd t h e g r e a t e s t n u m b e r 1203l of labelled neurons counted in t h i s experiment

other experiments, and labelled cells were found in t h e cerebral cortex in three of them (K7643, K7637, K7646).

K 7637 The peroxidase injection in K7637 was more caudally placed (fig. 1). It encompassed all but the rostral tip of t h e PO and heavily stained the lateral aspect of the MAO. The DAO was also well stained with HRP except for its rostral and caudal poles. Again, labelled cells were present in only a limited number of nuclei (table 4). The cerebral cortex contained many large heavily labelled cells, t h e vast majority of which were ipsilateral to the injection site. Several labelled neurons were again found around the ipsilateral caudal third ventricle and aqueduct in t h e subparafascicular nucleus, peri-ventricular gray, Nn. of Darkschewitsch, Cajal and adjacent reticular formation, and in t h e Edinger-Westphal nucleus. These cells were small to medium sized and contained a moderate amount of label. The pretectal complex was not labelled. Finally, the contralateral lateral cerebellar nucleus contained a few small, lightly labelled neurons in its medial hilus zone. The interpositus nuclei were not labelled, nor were t h e dorsal column nuclei and nucleus proprius of the spinal cord. K7642 In this experiment, the HRP injection was located more laterally (fig. 1). Except for i t s rostral tip, the PO was fairly well stained. The lateral aspect of the DAO was involved except for its rostral and caudal poles; however, only one small region in t h e lateral aspect of t h e MA0 was included in the injection site. The cerebral cortex was devoid of typically

labelled cells in this experiment (table 5 ) but, once again, t h e ipsilateral subparafascicular nucleus, peri-ventricular gray, Nn. of Darkschewitsch, Cajal and adjacent reticular formation, and the Edinger-Westphal nucleus contained several medium sized cells with moderate HRP labelling. The lateral cerebellar nucleus contained t h e greatest number of positive cells, most occurring contralateral to the injection site. Again, the majority of these cells were small, lightly labelled, and located in the medial hilus zone of the nucleus; however, some slightly larger positive neurons were found in the dorsal and caudolateral regions of t h e nucleus. Many small, variably labelled neurons were also seen in the lateral aspect of the contralateral interpositus nuclei. K7646 In animal K7646, a very limited peroxidase injection was made in the caudolateral part of the PO and DAO (fig. 1). Although the injection site was rather small, the locations of labelled neurons were more diverse in this experiment than in several of the others (table 6). K7646, therefore, helps confirm many origins of olivary afferents, particularly the pretectal complex and nucleus proprius of the spinal cord, found in t h e other studies. Since the locations, size, and HRP content of t h e labelled neurons in this experiment were similar to other experiments, a detailed description of them is unnecessary. DISCUSSION

Afferentsources rostral to the inferior olive Conflicting data a r e reported in the anatomic and physiologic literature concerning olivary afferents t h a t arise within the cerebral

9

AFFERENTS TO RAT INFERIOR OLIVE. H R P TABLE 5

Locations and incidence of labelled neurons following unilateral HRPinjection into rat inferior olive (K7642) Nucleus

lpsilateral

1. Subparafascicular nucleus 2. Peri-(third)ventricular gray 3. Nn. of Darkschewitsch, Cajal, and adjacent reticular formation 4. Edinger-Westphalnucleus 5. Lateral cerebellar nucleus 6. Interpositus nuclei of cerebellum

Midline

Contralateral

2+ 1+ 2+ 2+ 1+

31 2+

P e r t i n e n t negative findings-as in table 2 Plus m a r k s indicate t h e incidence of labelled neurons in each nucleus relative tu t h e contralateral lateral cerebellar nucleus which contained t h e g r e a t e s t n u m b e r 1681 of labelled neurons counted in t h i s experiment,

TABLE 6

Locations and incidence oflabelled neuronsfolloLL.rnRrlnilatera1HRPinjection into rat inferior olive (K7646) Nucleus

1. Cerebral cortex

2. Subparafascicular nucleus 3. Peri-(third)ventricular gray 4. Nn. of Darkschewitsch, Cajal, and adjacent reticular formation 5. Edinger-Westphalnucleus 6. Pretectal complex 7. Red nucleus 8. Lateral cerebellar nucleus 9. Interpositus nuclei of cerebellum 10. Medial cuneate nucleus 11. Gracile nucleus 12. N. proprius (spinal cord)

Ipsilateral

Midline

Contra lateral

1+ 2+ 1+

2+ 2+ 1+ 1+

1+

2 1 1+ l + 1+ 1+

PertinenL negative findings-as in table 2 P l u s m a r k s indicate t h e incidence of labelled neurons in each nucleus relative to t h e contralateral l a t e r a l cerebellar nucleus which contarned t h e g r e a t e s t n u m b e r (52) of labelled neurons counted in t h i s experiment.

cortex. Most of the anatomic studies in cats, in which degeneration techniques were employed, have shown a bilateral or predominantly contralateral cerebrocortical projection to the olive (Walberg, '56; Sousa-Pinto and Brodal, '69; Sousa-Pinto, '69). These olivary afferents were believed to originate mainly in the primary or secondary motor cortex and not in the sensory cortex (Sousa-Pinto and Brodal, '69; Sousa-Pinto, '69; Mizuno et al., '73b). Bishop et al. ('76b) injected HRP into the feline inferior olive and noted a bilateral projection from the motor cortex, less so from the sensory cortex. The present peroxidase study in the rat, however, in which a substantial, direct, predominantly ipsilatera1 sensorimotor projection to the inferior olive was found, is more in agreement with physiologic data (Armstrong and Harvey, '66; Provini et al., '68; Miller et al., '69a; Kitai et al., '69; Crill, '701, although not all cortico-olivary projections are necessarily monosyn-

aptic. See Allen and Tsukahara ('74) for a review. Sousa-Pinto and Brodal ('69) have suggested that the discrepancy between anatomic and physiologic data might be explained if some cortico-olivary projections had a relay in another nucleus like the globus pallidus. Walberg ('56) was able to demonstrate bilateral terminal degeneration in the olive after lesions in the pallidum and caudate, but other anatomic studies, including this one, have been unable to confirm olivary projections from either the pallidum (Stotler, '54; Nauta and Mehler, '66; Kim e t al., '76) or the caudate (Martin e t al., '75; Bishop et al., '76a). Although the ipsilateral sensorimotor cortex proved to be a rich source of olivary afferents, we were unable to find any labelled cells in the rat visual and auditory cortices. This agrees with the data of Bishop et al. ('76b) and Martin et al. ('75). A great number of anatomic studies, largely employing anterograde degeneration tech-

10

J. T. BROWN, V. CHAN-PALAY AND S. L. PALAY

niques, have consistently demonstrated a n olivary projection from t h e meso-diencephalic junction around the third ventricle and aqueduct. That many of the lesions were quite large and may have damaged fibers of passage as well a s cell bodies has made identification of t h e exact origin of this projection difficult. Nonetheless, in 1939, Ogawa was able to demonstrate, in the cat, connections from the N. of Darkschewitsch, the interstitial N. of Cajal and the N. of Forel's field to the ipsilateral inferior olive, and Mettler ('44), similarly employing t h e Marchi method, illustrated a n "annulo-olivary" tract in the monkey originating in t h e ventral and lateral periaqueductal gray and possibly the EdingerWestphal nucleus. In extensive silver degeneration studies, Walberg ('56, '60, '74) showed a dense projection from the ventral periaqueductal gray, particularly t h e N. of Darkschewitsch, and from the adjacent reticu l a r formation to t h e ipsilateral olive. Mabuchi and Kusama ('70), using silver techniques in the cat, demonstrated t h a t lesions involving the N. of Darkschewitsch and t h e interstitial N. of Cajal and extending rostrally into t h e tegmental field of Fore1 and caudal thalamus would yield degeneration products in t h e ipsilateral olive. Other a n a tomic and physiologic studies, primarily in the cat and monkey (Miller e t al., '69b; Hamilton and Skultety, '70; Carpenter et al., '70), have confirmed this largely ipsilateral projection to t h e inferior olive. I t has only been with t h e development of t h e peroxidase technique, however, t h a t t h e details of this annulo-olivary connection have begun to be revealed. Henkel et al. ('751, in t h e opossum, were t h e first to apply t h e H R P technique for this purpose, and, after olivary injections, they noted labelled cells ipsilaterally in t h e subparafascicular nucleus and a d j a c e n t c e n t r a l gray, t h e N. of D a r k schewitsch, t h e interstitial N. of Cajal, Forel's field and the midbrain tegmentum, and bilaterally in the ventral and lateral peri-aqueductal gray, the nucleus of the posterior commissure and in the deep and lateral midbrain tegmentum. Their injection sites were quite large, however, and spread well beyond t h e confines of t h e inferior olive. Although t h e precision of their injections is unknown, Linauts and Martin ('76) in the opossum and Bishop e t al. ('76a) in t h e cat have recently used the same method to ascertain olivary

afferents and have obtained results quite similar to those of Henkel et al. ('75). W i t h a few exceptions, the present work, in which only labelling from well-localized injection sites was studied, agrees with the previous authors. The major difference is that according to the present results in t h e r a t , the annulo-olivary projection is exclusively ipsilateral and consistently provides one of the richest sources of olivary afferents. Furthermore, the Edinger-Westphal nucleus is shown to contain numerous labelled cells. Mettler ('44) indicated t h a t the Edinger-Westphal nucleus might project to the ipsilateral olive, and Walberg ('74) could not rule out the possibility that this nucleus, as well as the N. of Darkschewitsch, provides olivary afferents, since both were included in lesions of the mesencephalic central gray. In a cerebellar autoradiographic study in r a t and monkey, Chan-Palay ('77) confirmed some of the earlier results of Carpenter and Strominger ('64) by demonstrating a predominantly contralateral projection from t h e lateral cerebellar nucleus to not only the midbrain eye centers for vertical and rotatory gaze, the N. of Darkschewitsch and t h e interstitial N. of Cajal, but also t h e oculomotor nucleus and Edinger-Westphal nucleus. This pathway has been implicated in the coordination, if not initiation, of certain eye movements such a s saccades (Ron and Robinson, '73; Aschoff, '74; Gardner and Fuchs, '75). For a review, see Chan-Palay ('77). Considering the olivary projection to t h e lateral cerebellar nucleus as determined in t h e rat by Eller and ChanPalay ('76) and in the monkey by Chan-Palay ('771, it would appear t h a t these accessory oculomotor and autonomic eye centers are involved in a closed loop feedback system, via t h e olive, with t h e lateral cerebellar nucleus, and this system could possibly be of importance in cerebellar influence on eye movement. The rostra1 red nucleus has long been thought to provide a major input to the ipsilateral inferior olive (Walberg, '56; Hinman and Carpenter, '59; Edwards, '72; Courville and Otabe, '73; Miller and Strominger, '73; Martin et al., '75). In fact, Stotler ('54) found degeneration in t h e olive of t h e cat only if the ipsilateral red nucleus was destroyed, despite lesions in several other meso-diencephalic structures, including t h e central gray. I t was therefore a surprise to find relatively

A F F E R E N T S TO RAT INFERIOR OLIVE, HKP

few labelled neurons in t h e ipsilateral red nucleus of the rat. Henkel e t al. ('751, however, also reported very few labelled neurons in the red nucleus using t h e peroxidase technique in the opossum, and Bishop e t al. ('76a) do not even mention the red nucleus in their list of labelled regions in t h e cat. Although extreme caution must be exercised in the interpretation of negative or marginal results with the HRP method (Nauta e t al., '741, i t is possible t h a t the rubro-olivary tract is not as prodigious a s was once thought, at least in lower mammals, and t h a t previous demonstrations of supposedly extensive rubro-olivary connections in these animals by means of degeneration techniques might be due to interruption of fibers of passage. The pretectal complex is t h e final significant source of afferents which arise rostra1 to the inferior olive. There is general agreement t h a t the retinal projection to t h e pretectal complex is primarily crossed, and degeneration experiments following eye removal have, on the whole, shown retinal projections to the contralateral N. of t h e optic tract, olivary pretectal N., and anterior and posterior pretectal Nn., with a smaller contingent of fibers terminating in t h e ipsilateral complex (Singleton and Peele, '65; Laties and Sprague, '66; Giolli and Guthrie, '69; Scalia, '72). I n a physiologic study, Siminoff et al. ('67) recorded evoked potentials in t h e anterior pretectal nucleus after stimulating t h e contralateral retina with light. There have been few attempts to decipher the nature of pretecto-olivary connections, however. Graybiel ('741, employing a n autoradiographic method in the cat, showed t h a t such a pathway exists, and Mizuno e t al. ('73a1, using degeneration techniques in rabbits, demonstrated t h a t the pretectal area projects to the ipsilateral dorsal cap and nucleus p of t h e inferior olive. In a recent physiologic study, Hoffmann e t al. ('76) provided evidence for a monosynaptic connection between the nucleus of t h e optic tract and the ipsilateral feline inferior olive. Only with the peroxidase method, however, can it best be shown precisely which pretectal nuclei send axons to t h e olive, and t h e present study in the rat demonstrates a strictly ipsilateral projection to the olive originating in t h e N. of the optic tract, olivary pretectal N., posterior pretectal N., and, especially, in the anterior pretectal N. The vestibulo-ocular reflex serves to stabil-

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ize retinal images by producing eye movement compensatory for head movement. For a review, see Chan-Palay ('77). Details of the neuronal connections of this reflex and its influence by the vestibulo-cerebellum have recently been worked out in the rabbit by Highstein and Ito and their co-workers (Highstein, '73a,b; Ito et al., '73). As part of his hypothesis explaining how t h e cerebellum might participate in t h e adaptive control of the vestibulo-ocular reflex, Ito ('72) proposed a visual feedback to t h e cerebellum via the inferior olive. Maekawa and Simpson ('73) then physiologically traced a pathway from t h e retina to the contralateral accessory optic tract and olive and back to the ipsilateral vestibulo-cerebellum via olivary climbing fibers. They proposed a synapse for the crossed retinal fibers in t h e lateral pretectum and/or dorsal terminal nucleus, the axons of which would then project monosynaptically to the inferior olive of the same side (fig. 2). Although retinal fibers distribute to the contralateral pretectal complex, current data indicate t h a t they arrive via collaterals of fibers coursing within the brachium of the superior colliculus (Giolli and Guthrie, '691, and not via the accessory optic tract. The latter tract has been shown in various mammals to be a separate, distinct, and totally crossed pathway ending in the dorsal, lateral and, particularly, t h e medial terminal accessory optic nuclei and not in t h e pretectal complex (Hayhow, '59; Hayhow et al., '60; Laties and Sprague, '66; Giolli and Guthrie, '69). Our findings indicate t h a t t h e entire pretectal complex, particularly t h e caudolateral part of the anterior pretectal N., projects to t h e ipsilateral olive, but no labelled cells were found in t h e terminal nuclei of the accessory optic tract. The pretecto-olivary connection demonstrated in t h e present study, therefore, provides a more precise link in a pathway whereby visual information might gain access to t h e vestibulo-cerebellum to influence cerebellar modulation of t h e vestibulo-ocular reflex.

Afferent sources caudal to the inferior olive Spinal input to the olive is both direct and indirect. The indirect route is believed to involve a relay at least in the dorsal column nuclei. Spinal afferents traveling primarily in the ventral funiculus of t h e cord have been shown by various degeneration methods to

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J . T. BROWN. V. CHAN-PALAY AND S. L. PALAY

terminate predominantly in t h e ipsilateral olive of the monkey (Mehler et al., '60; Mehler, '69), c a t (Brodal et al., '50; Mizuno, '66; Mehler, '69; Boesten and Voogd, '75), pig (Breazile and Kitchell, '681, r a t (Mehler, '691, rabbit (Mizuno, '66; Mehler, '691, opossum (Mehler, '69; Hazlett e t al., '72; Martin et al., '75), hedgehog (Jane and Schroeder, '71) and tree shrew (Schroeder and Jane, '71). The dorsal accessory and caudal medial accessory olives have been shown by Brodal e t al. ('50) and Mizuno ('66) to be the major olivary targets of afferents originating in t h e spinal cord, and these investigators also supposed t h a t the cells of origin within the cord were located in the dorsal horn mainly contralateral to the ventral funiculus and olive under study. The present peroxidase study, although i t does not allow delineation of t h e spino-olivary tract within the cord or t h e exact area of termination within the olive, does demonstrate t h a t , in the rat, the cells of origin lie in the medial aspect of the nucleus proprius contralateral to the injected olive. Although numerous sections from all cord levels were examined, no labelled cells were found below (2-4. This is probably related to the short survival time after injection since the degeneration studies have adequately shown the spino-olivary tract to arise from all levels of t h e spinal cord. A direct spino-olivary projection in the r a t t h a t originates in the nucleus proprius, crosses, probably in t h e anterior white commissure, and ascends in t h e ventral funiculus to t h e contralateral inferior olive is therefore postulated. Although Brodal e t al. ('50) found no degeneration in the inferior olive of the cat after lesions in the dorsal column nuclei, several feline degeneration a n d autoradiographic studies since t h a t time have shown t h a t t h e dorsal column nuclei are intimately related to the contralateral dorsal and medial accessory olives (Morest, '67; Ebbesson, '68; Boesten and Voogd, '75; Groenewegen et al., '75). Silver methods applied to t h e tree shrew (Schroeder and Jane, '711, hedgehog (Jane and Schroeder, '711, and opossum (Hazlett et al., '72), however, have concluded t h a t both dorsal column nuclei project only to t h e contralateral dorsal accessory olive. The present experiments in the r a t show t h a t both dorsal column nuclei send axons to the contralateral inferior olive, but precisely where within the olive these axons terminate cannot be stated.

Our peroxidase results are similar to those of Bishop et al. ('76a). We have added further evidence t h a t the dorsal column nuclei are connected monosynaptically with the contralateral inferior olive and thus can serve as a relay in a n indirect spino-olivary tract. Oscarsson has physiologically demonstrated spino-olivo-cerebellar pathways t h a t course in the ventral and dorsal funiculi of t h e spinal cord (Oscarsson, '68, '69). The ventral funiculus spino-olivo-cerebellar pathway (VF-SOCP) mediates impulses from flexor reflex afferents in all four limbs but mainly from limbs ipsilateral to the side of cerebellar recording. The pathway probably involves neurons in t h e ipsilateral central gray of the cord whose axons cross to t h e contralateral ventral funiculus and ascend to the olive, where a relay projects the information back to the ipsilateral cerebellum (fig. 3). Inputs from limbs contralateral to t h e side of cerebellar recording possibly reach the ipsilateral central gray via interneurons. I t is proposed that t h e nucleus proprius neurons labelled in this study are t h e immediate relay from the cord to the contralateral olive in the VF-SOCP. The dorsal funiculus spino-olivo-cerebellar pathway (DF-SOCP),on the other hand, mediates input from flexor reflex afferents located only in limbs ipsilateral to t h e side of cerebellar recording. The pathway is disynaptic and involves axons which ascend in the ipsilateral dorsal funiculus, relay in t h e dorsal column nuclei and again in t h e contralateral olive, from which, the final projection is back to the ipsilateral cerebellum (fig. 3). The crossed relationship of gracilo-olivary and cuneato-olivary fibers amply demonstrated here and in other studies thus provides a link in the DFSOCP. Larson e t al. ('69a,b) proposed two additional indirect spinal pathways t h a t mediate information from limb afferents to the cerebellum via climbing fibers. The dorsolateral funiculus spino-olivo-cerebellar pathway (DLF-SOCP) t r a n s m i t s information from cutaneous afferents in limbs ipsilateral to the side of cerebellar recording. After a possible synapse in t h e cord, t h e pathway ascends in t h e dorsal part of t h e ipsilateral lateral funiculus to t h e brainstem where i t traverses several synapses before arriving at the contralateral olive and thence the ipsilateral cerebellum (fig. 4). The lateral funiculus-climbing fiber-spino-cerebellar pathway (LF-CF-

AFFERENTS TO RAT INFERIOR OLIVE, HRP

Ketino

- Pretecto - Olivo - Cerebellar

Pathway

pathway. Retinal input to the ipsilateral cerebellum is relayed in the Fig. 2 Retino-pretecto-olivo-cerebellar contralateral pretectal complex and inferior olive. The pretecto-olivary pathway is uncrossed.

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J T BROWN V CHANPALAY AND S L PALAY

V e n t r a l F u n i c u l u s S p i n o - Olivo- C e r e b e l Iar Pathway (VF-SOCP)

-- - -

---

I > o r s a 1 F u n I cu l u s Spino- 0 1 i v o - C e r e be1 l u r P a t h w a y (DF- S O c P)

Fig. 3 Ventral funiculus and dorsal funiculus spino-olivo-cerebellar pathways. Spinal input to the ipsilateral cerebellum via t h e VF-SOCP (dashed line1 relays in t h e ipsilateral nucleus proprius of t h e cord and in the contralateral olive. Spinal input to t h e ipsilateral cerebellum via t h e DF-SOCP (continuous line1 relays in t he ipsilateral dorsal column nuclei and contralateral olive.

SCP), which can now properly be called t h e lateral funiculus spino-olivo-cerebellar pathway (LF-SOCP) since Armstrong et al. ('73) showed a definite relay in the olive, mediates information from flexor reflex afferents of all four limbs. Impulses from limbs ipsilateral to the side of cerebellar recording probably relay in t h e spinal central gray of t h a t side, while impulses from the contralateral side arrive

via spinal interneurons. Axons then ascend in t h e ventral part of t h e ipsilateral lateral funiculus to the brainstem where, again, the pathway probably crosses several synapses on t h e way to t h e contralateral olive and from there to the ipsilateral cerebellum (fig. 4).Although several contemporary studies have dealt with efferent projections from the lateral reticular nucleus (LRN) to the cerebel-

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AFFERENTS TO RAT INFERIOR OLIVE, HRP

D o r s o l a t e r a l F u n i c u l u s S p i n o - O l i v o - (:crct)cl l a r P a r h w s y ( I > L , F - S O C 1') - - - - - -

L a t e r a l ~u n icu 111 s

s p i n o - o I i v o - .. S Hedman and P S t r a t a 1968 Mossy and c,limhing fihre organization on the anterior lobe of t h e cerebellum activ'ited by forelimb and hindlimb areas of the sensorimotor cortex. Exp. Brain Res , 6. 216-233. Rand. R N' 1954 An anatomical and experimental %tudyof t h e cerebellar nuclei and their efferent p a t h ways in the monkey J. Comp Neur.. 101: 167-223. Ron. S , and D A Robinson 1973 Eye movements evoked by cerebellar stimulation i n t h e alert monkey J Neurophysiol , 36 1004-1022 Scalia. F 1972 The termination of retinal axons in t h e pre tectal region of mammals J Comp. Neur , 145 223"8

Schroeder. D M , a d J A. J a n e 1971 Projection of dorsal column nuclei a n d spinal cord t o brainstem and thalamus i n t h e tree shrew. Tupain glis J Comp Neur , 142: 309350 Sherlock. D A.. a n d G Raisman 1975 A comparison of a n teroprade and retrograde nxonul transport of horseradish peroxidase in t h e connections of the mammillary nuclei i n t h r r a t Brain Res , 85. 321 324.

Sidman. R. L , J . B. Angevine and E. T. Pierce 1971 Atlas of t h e Mouse Brain a n d Spinal Cord. Harvard University Press. Cambridge. Massachusetts. SiminufT, R.. H. 0 Schwassmann and L. Kruger 1967 Unit analysis of t h e pretectal nuclear group in t h e rat J. Comp. Neur.. 130 329.342. Singleton, M C.. and T L. Peele 1965 Distribution of optic fibers in t h e cat J . Comp. Neur , 125 303-328. Sladek. J. R.. a n d J. P. Bowman 1975 The distribution of catecholamines within the inferior olivary complex of the cat and Rhesus monkey. J . Comp. Neur , 163; 203-214. Sousa-Pinto, A. 1969 Experimental anatomical demonstration of a cortico-olivary projection from area 6 in cat. Brain Res.. 16 73-83. Sousa-Pinto. A , . and A. Brodal 1969 Demonstration of a somatotopical pattern in t h e cortico-olivary projection in the cat. An experimental anatomical study. Exp. Brain Res . 8: 364-386. Stotler. W. A . 1954 An experimental study of the origin of the afferent fibers of the inferior olivary nucleus of the cat brain. A n a t . R e c , 118: 359 ( A b s t r a c t ) . Szentagothai, J., a n d K. Rajkovits 1959 Uher den Ursprung der Kletterfasern des Kleinhirns. Z. A n a t Ent. wickl.-Gesch.. 121. 130-141. Tolbert. D L.. L. C. Massopust. M. G . Murphy a n d P A. Young 1976 The anatomical organization of the cerebel. lo-olivary projection in the c a t . J. Comp. Neur.. I70 525. 544. Walberg. F 1956 Descending connections t o t h e inferior olive. An experimental study in t h e cat. J. Comp. Neur , 104 77.173 1960 F u r t h e r studies on t h e descending connections to t h e inferior olive: Reticulo-olivary fibers. An ex perimental study in the cat. J Comp. Neur.. 114 79-87 1974 Descending connections from t h e mesencephalon t o t h e inferior olive. An experimental study in the cat Exp. Brain Res.. 20: 145-156. Wolfe. J . W. 1971 Relationship of cerebellar potentials t o saccadic eye movements. Brain Res.. 30. 204-206.

A study of afferent input to the inferior olivary complex in the rat by retrograde axonal transport of horseradish peroxidase.

A Study of Afferent Input to the Inferior Olivary Complex in the Rat by Retrograde Axonal Transport of Horseradish Peroxidase JAMES T. BROWN,' VICTORI...
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