Brain Research, 85 (1975) 321-324
© ElsevierScientificPublishing Company, Amsterdam - Printed in The Netherlands
321
A COMPARISON OF ANTEROGRADE AND RETROGRADE AXONAL TRANSPORT OF HORSERADISH PEROXIDASE IN THE CONNECTIONS OF THE MAMMILLARY NUCLEI IN THE RAT
D. A. S H E R L O C K
AND G. R A I S M A N
University of Oxford, Department of Human Anatomy, South Parks Road, Oxford OX1 3QX (Great Britain)
Since several of the efferent and afferent fibre connections of the mammillary nuclei are well characterizedl,a-5,7, a single injection of horseradish peroxidase into the mammillary complex allows anterograde 11 and retrograde 9,1°,12 axonal transport of horseradish peroxidase to be studied simultaneously. Major inputs to the mammillary nuclei are from the hippocampus via the fornix, and from the deep and dorsal tegmental nuclei via the mammillary peduncle. The axons of the principal mammillary efferent tract bifurcate to form the mammillothalamic tract, which projects to the anterior group of thalamic nuclei, and the mammillotegmental tract which projects principally to the deep and dorsal tegmental nuclei. Twenty-one female Wistar rats of approximately 200 g weight were used. 0.1 #1 (or, in a few cases, as much as 1.1/zl) of a 20-60 ~ solution of horseradish peroxidase (HRP, Sigma Types II or IV), dissolved in distilled water, was injected over a period of 4-9 min through a glass micropipette of tip diameter approximately 60 #m located stereotaxically in the left mammillary complex. After injection, the system was allowed to equilibrate for a further 10 min, before withdrawing the pipette. The animals were allowed to survive for periods ranging from 20 h to 30 days before being anaesthetized and perfused transcardially with 1 litre of a mixture of either 1 ~ or 3 ~ formaldehyde and 1 ~ glutaraldehyde in 0.05 M phosphate buffer (pH 7.4). The brain was then removed from the skull and placed in fixative for 4-6 h, after which it was washed overnight in 0.05 M phosphate buffer containing 5 ~ sucrose (PBS) and 1 ~ dimethylsulphoxide (DMSO), pH 7.4. The brain was frozen and serial coronal or sagittal sections, 40-50 #m thick, were cut into a solution of 50 mg of 3,3'-diaminobenzidine hydrochloride (DAB, Sigma)6,10 in 100 ml of PBS and 1 ~o DMSO and preincubated for 30 min at room temperature. The sections were next transferred to a solution of 50 mg of DAB in 100 ml of PBS and 1 ~ DMSO to which had been added 3.5 ml of 0.3 ~ hydrogen peroxide, and incubated for 30 min. This was followed by 3 washes of PBS and 1 wash of distilled water before the sections were mounted in 0.5 gelatin in 40 ~ alcohol. Sections both unstained, and stained with cresyl violet, were viewed using dark-field and bright-field illumination. In the subsequent account the observations of both retrograde and anterograde labelling were on the same side
322 as that of the injection, and no label was seen on the contralateral (control) side. As an additional control procedure, some sections were treated with DAB alone (i.e. without hydrogen peroxide being present at any stage). Further controls were provided by processing to demonstrate peroxidase using unoperated rats and also rats with radiofrequency lesions in the mammillary nuclei: in neither case was HRP reaction product present. Injection sites (Fig. 1) were visible macroscopically as dark brown loci of varying size. Microscopically, injection of 0.1 #1 of HRP produced a lesion of between 200 and 400 # m diameter which destroyed up to one-third of the mammillary nucleus. Spread of HRP occurred as 'tracking' back along the path of the pipette through the brain, and also as a sphere of about 1-1.5 mm diameter around the lesion. The size of the spread of HRP was progressively reduced the longer the animal survived and at 7 days only intracellular labelling, not visible macroscopically, was present. Free H R P was present at early survival times, giving rise to a uniform deep golden brown stain which lightened towards the limits of the injection site. Reaction product was occasionally seen in neurones, staining evenly, and as granules in presumed phagocytes. Pericytes of blood vessels showed cytoplasmic granules for some distance from the lesion. A few axons near the pipette path stained evenly, but providing they were not directly damaged, bundles of axons such as the habenulo-interpeduncular tract, although passing very close to the pipette, did not show significant H R P uptake s. Endogenous reaction product was seen in all operated and unoperated animals in the region of the arcuate nucleus of the hypothalamus, around the posterior end of the third ventricle and in the area postrema of the brain stem. Axons in the mammillary peduncle showed even staining (Fig. 3) as they pass posteriorly along the base of the brain. A few labelled axons continued dorsally towards the deep tegmental nucleus. Using Type VI H RP, cells of the deep tegmental nucleus showed many large granules of reaction product in their cytoplasm (Fig. 4), thus demonstrating retrograde labelling. In a few cases, labelled neurones were seen in the vicinity of the dorsal tegmental nucleus. In all except one case, no label was seen in the fornix, and in no case in the hippocampal pyramidal cells. HRP also appeared to be transported orthogradely in the principal mammillary tract, and effectively outlined the bifurcation of this tract (Fig. 2) into mammillothalamic and mammillotegmental tracts, described by Cajal 2. Reaction product was seen in the region of the terminal distribution of the mammillothalamic tract in the anterior group of thalamic nuclei. No label could be seen in the terminal region of the mammillotegmental tract. These experiments show that at the same survival time, both retrograde and orthograde axonal transport of H R P can occur from the same injection site, and that at light microscopic levels axons transporting HRP orthogradely cannot be distinguished from axons transporting HRP retrogradely. In order to distinguish retrograde from orthograde axonal HRP labelling, it is necessary to demonstrate the connections of the axons either with cells of origin or with a terminal field. Inspection of the injection sites in the mammillary nuclei shows that both retrograde and orthograde transport of H R P seems to be greater in cases where a
323
Fig. 1. A sagittal section through the injection site showing pipette track (arrow) and reaction product in the surrounding tissue. Fig. 2. A sagittal section showing the bifurcation of the labelled axons of the principal mammillary efferent tract (P) into the mammillothalamic (T) and mammillotegmental tracts (G). Fig. 3. A sagittal section through the mammillary peduncle at a level where the ascending axons form a compact, labelled bundle along the base of the brain just caudal to the mammillary nuclei. Fig. 4. A sagittal section showing heavily labelled neurones in the deep tegmental nucleus. All photomicrographs were taken using dark ground illumination. Calibration bar = 100 ~m for all figures.
lesion is produced. In some cases the principal mammillary efferent tract was totally transected by a lesion resulting from injections of larger volumes of H R P solution. In these cases the observed orthograde transport of H R P in the mammillothalamic tract must represent material taken up by, and flowing down, degenerating axons completely severed from their cell bodies. In our system the deep tegmental nucleus shows marked retrograde H R P transport; this is interesting because, as was shown by Akert and Andy 1, and Cowan et al. 3, the deep tegmental nucleus shows marked retrograde cellular degeneration following mammillary lesions. We have not been able to detect major cellular labelling
324 in the dorsal tegmental nucleus, which shows only m i n o r r e t r o g r a d e cellular degeneration. The p y r a m i d a l cells o f the h i p p o c a m p u s show no r e t r o g r a d e changes alter m a m m i l l a r y lesions 4 a n d show no trace o f r e t r o g r a d e a x o n a l t r a n s p o r t o f HRP. Two m a i n differences were observed between the b e h a v i o u r o f Types 11 and VI H R P . T r a n s p o r t o f T y p e 1I H R P was n o t seen before 1 week after o p e r a t i o n , it reached a m a x i m u m at 2 weeks and h a d d i s a p p e a r e d by 3 weeks. In contrast, t r a n s p o r t o f T y p e VI H R P was seen as early as 1 d a y after o p e r a t i o n , was very m a r k e d at 2 days a n d had d i s a p p e a r e d by 1 week. The second difference involved the d i s t r i b u t i o n o f r e t r o g r a d e t r a n s p o r t . R e t r o g r a d e t r a n s p o r t o f T y p e 1I H R P was seen in the m a m m i l l a ry peduncle, b u t d i d n o t satisfactorily label the cells o f origin in the deep tegmental nuclei (at least at light m i c r o s c o p i c levels). T y p e VI H R P labelled not only the axons in the m a m m i l l a r y p e d u n c l e but also the cells o f the deep tegmental nuclei and in one case even the distal p a r t o f the fornix c o l u m n . T y p e 11 H R P is a less active, less highly purified form than T y p e VI, a n d these differences m a y be due to variation in the effective c o n c e n t r a t i o n o f H R P at the site o f injection.
1 AKERT, K., AND ANDY, O. J., Experimental studies on corpus mammillare and tegmento-mammillary system in the cat, Amer. J. Physiol., 183 (1955) 591. 2 CAJAL, S. RAMON Y, Histologie du SystOme Nerveux de L'Homme et des Vert~brds, Vol. 11,
Maloine, Paris, 1911, pp. 456-474. 3 COWAN, W. M., GUILLERY, R. W., AND POWELL, T. P. S., The origin of the mammillary peduncle and other hypothalamic connexions from the midbrain, J. Anat. (Lond.), 98 0964) 345-363. 4 DA1TZ, H. M., AND POWELL, T. P. S., Studies of the connexions of the fornix system, J. Neurol. Psychiat., 17 (1954) 75-82.
5 FRY, W. J., Quantitative delineation of the efferent anatomy of the medial mammillary nucleus of the cat, J. comp. NeuroL, 139 (1970) 321-336. 6 GRAHAM, R. C., JR., AND KARNOVSKY, M. J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. 7 GUILLERY, R. W., Degeneration in the post-commissural fornix and the mamillary peduncle of the rat, J. Anat. (Lond.), 90 (1956) 350-370. 8 KRISHNAN, N., AND SINGER, M., Penetration of peroxidase into peripheral nerve fibers, Amer. J. Anat., 136 (1973) 1-14. 9 KRISTENSSON,K., OLSSON, Y., AND SJ6STRAND, J., Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve, Brain Research, 32 (1971) 399-406. 10 LAVAIL, J. H., WINSTON, K. R., AND TISH, A., A method based on retrograde intraaxonal transport of protein for identification of cell bodies of origin of axons terminating within the CNS, Brain Research, 58 (1973) 470-477. 11 LYNCH,G., GALL, C., MENSAH,P., AND COTMAN,C. W., Horseradish peroxidase histochemistry: a new method for tracing efferent projections in the central nervous system, Brain Research, 65 (1974) 373-380. 12 NAUTA, H. J. W., PRITZ, M. B., AND LASEK, R. J., Afferents to the rat caudoputamen studied with horseradish peroxidase. An evaluation of a retrograde neuroanatomical research method, Brain Research, 67 (1974) 219-238.
© ElsevierScientificPublishing Company, Amsterdam - Printed in The Netherlands
321
A COMPARISON OF ANTEROGRADE AND RETROGRADE AXONAL TRANSPORT OF HORSERADISH PEROXIDASE IN THE CONNECTIONS OF THE MAMMILLARY NUCLEI IN THE RAT
D. A. S H E R L O C K
AND G. R A I S M A N
University of Oxford, Department of Human Anatomy, South Parks Road, Oxford OX1 3QX (Great Britain)
Since several of the efferent and afferent fibre connections of the mammillary nuclei are well characterizedl,a-5,7, a single injection of horseradish peroxidase into the mammillary complex allows anterograde 11 and retrograde 9,1°,12 axonal transport of horseradish peroxidase to be studied simultaneously. Major inputs to the mammillary nuclei are from the hippocampus via the fornix, and from the deep and dorsal tegmental nuclei via the mammillary peduncle. The axons of the principal mammillary efferent tract bifurcate to form the mammillothalamic tract, which projects to the anterior group of thalamic nuclei, and the mammillotegmental tract which projects principally to the deep and dorsal tegmental nuclei. Twenty-one female Wistar rats of approximately 200 g weight were used. 0.1 #1 (or, in a few cases, as much as 1.1/zl) of a 20-60 ~ solution of horseradish peroxidase (HRP, Sigma Types II or IV), dissolved in distilled water, was injected over a period of 4-9 min through a glass micropipette of tip diameter approximately 60 #m located stereotaxically in the left mammillary complex. After injection, the system was allowed to equilibrate for a further 10 min, before withdrawing the pipette. The animals were allowed to survive for periods ranging from 20 h to 30 days before being anaesthetized and perfused transcardially with 1 litre of a mixture of either 1 ~ or 3 ~ formaldehyde and 1 ~ glutaraldehyde in 0.05 M phosphate buffer (pH 7.4). The brain was then removed from the skull and placed in fixative for 4-6 h, after which it was washed overnight in 0.05 M phosphate buffer containing 5 ~ sucrose (PBS) and 1 ~ dimethylsulphoxide (DMSO), pH 7.4. The brain was frozen and serial coronal or sagittal sections, 40-50 #m thick, were cut into a solution of 50 mg of 3,3'-diaminobenzidine hydrochloride (DAB, Sigma)6,10 in 100 ml of PBS and 1 ~o DMSO and preincubated for 30 min at room temperature. The sections were next transferred to a solution of 50 mg of DAB in 100 ml of PBS and 1 ~ DMSO to which had been added 3.5 ml of 0.3 ~ hydrogen peroxide, and incubated for 30 min. This was followed by 3 washes of PBS and 1 wash of distilled water before the sections were mounted in 0.5 gelatin in 40 ~ alcohol. Sections both unstained, and stained with cresyl violet, were viewed using dark-field and bright-field illumination. In the subsequent account the observations of both retrograde and anterograde labelling were on the same side
322 as that of the injection, and no label was seen on the contralateral (control) side. As an additional control procedure, some sections were treated with DAB alone (i.e. without hydrogen peroxide being present at any stage). Further controls were provided by processing to demonstrate peroxidase using unoperated rats and also rats with radiofrequency lesions in the mammillary nuclei: in neither case was HRP reaction product present. Injection sites (Fig. 1) were visible macroscopically as dark brown loci of varying size. Microscopically, injection of 0.1 #1 of HRP produced a lesion of between 200 and 400 # m diameter which destroyed up to one-third of the mammillary nucleus. Spread of HRP occurred as 'tracking' back along the path of the pipette through the brain, and also as a sphere of about 1-1.5 mm diameter around the lesion. The size of the spread of HRP was progressively reduced the longer the animal survived and at 7 days only intracellular labelling, not visible macroscopically, was present. Free H R P was present at early survival times, giving rise to a uniform deep golden brown stain which lightened towards the limits of the injection site. Reaction product was occasionally seen in neurones, staining evenly, and as granules in presumed phagocytes. Pericytes of blood vessels showed cytoplasmic granules for some distance from the lesion. A few axons near the pipette path stained evenly, but providing they were not directly damaged, bundles of axons such as the habenulo-interpeduncular tract, although passing very close to the pipette, did not show significant H R P uptake s. Endogenous reaction product was seen in all operated and unoperated animals in the region of the arcuate nucleus of the hypothalamus, around the posterior end of the third ventricle and in the area postrema of the brain stem. Axons in the mammillary peduncle showed even staining (Fig. 3) as they pass posteriorly along the base of the brain. A few labelled axons continued dorsally towards the deep tegmental nucleus. Using Type VI H RP, cells of the deep tegmental nucleus showed many large granules of reaction product in their cytoplasm (Fig. 4), thus demonstrating retrograde labelling. In a few cases, labelled neurones were seen in the vicinity of the dorsal tegmental nucleus. In all except one case, no label was seen in the fornix, and in no case in the hippocampal pyramidal cells. HRP also appeared to be transported orthogradely in the principal mammillary tract, and effectively outlined the bifurcation of this tract (Fig. 2) into mammillothalamic and mammillotegmental tracts, described by Cajal 2. Reaction product was seen in the region of the terminal distribution of the mammillothalamic tract in the anterior group of thalamic nuclei. No label could be seen in the terminal region of the mammillotegmental tract. These experiments show that at the same survival time, both retrograde and orthograde axonal transport of H R P can occur from the same injection site, and that at light microscopic levels axons transporting HRP orthogradely cannot be distinguished from axons transporting HRP retrogradely. In order to distinguish retrograde from orthograde axonal HRP labelling, it is necessary to demonstrate the connections of the axons either with cells of origin or with a terminal field. Inspection of the injection sites in the mammillary nuclei shows that both retrograde and orthograde transport of H R P seems to be greater in cases where a
323
Fig. 1. A sagittal section through the injection site showing pipette track (arrow) and reaction product in the surrounding tissue. Fig. 2. A sagittal section showing the bifurcation of the labelled axons of the principal mammillary efferent tract (P) into the mammillothalamic (T) and mammillotegmental tracts (G). Fig. 3. A sagittal section through the mammillary peduncle at a level where the ascending axons form a compact, labelled bundle along the base of the brain just caudal to the mammillary nuclei. Fig. 4. A sagittal section showing heavily labelled neurones in the deep tegmental nucleus. All photomicrographs were taken using dark ground illumination. Calibration bar = 100 ~m for all figures.
lesion is produced. In some cases the principal mammillary efferent tract was totally transected by a lesion resulting from injections of larger volumes of H R P solution. In these cases the observed orthograde transport of H R P in the mammillothalamic tract must represent material taken up by, and flowing down, degenerating axons completely severed from their cell bodies. In our system the deep tegmental nucleus shows marked retrograde H R P transport; this is interesting because, as was shown by Akert and Andy 1, and Cowan et al. 3, the deep tegmental nucleus shows marked retrograde cellular degeneration following mammillary lesions. We have not been able to detect major cellular labelling
324 in the dorsal tegmental nucleus, which shows only m i n o r r e t r o g r a d e cellular degeneration. The p y r a m i d a l cells o f the h i p p o c a m p u s show no r e t r o g r a d e changes alter m a m m i l l a r y lesions 4 a n d show no trace o f r e t r o g r a d e a x o n a l t r a n s p o r t o f HRP. Two m a i n differences were observed between the b e h a v i o u r o f Types 11 and VI H R P . T r a n s p o r t o f T y p e 1I H R P was n o t seen before 1 week after o p e r a t i o n , it reached a m a x i m u m at 2 weeks and h a d d i s a p p e a r e d by 3 weeks. In contrast, t r a n s p o r t o f T y p e VI H R P was seen as early as 1 d a y after o p e r a t i o n , was very m a r k e d at 2 days a n d had d i s a p p e a r e d by 1 week. The second difference involved the d i s t r i b u t i o n o f r e t r o g r a d e t r a n s p o r t . R e t r o g r a d e t r a n s p o r t o f T y p e 1I H R P was seen in the m a m m i l l a ry peduncle, b u t d i d n o t satisfactorily label the cells o f origin in the deep tegmental nuclei (at least at light m i c r o s c o p i c levels). T y p e VI H R P labelled not only the axons in the m a m m i l l a r y p e d u n c l e but also the cells o f the deep tegmental nuclei and in one case even the distal p a r t o f the fornix c o l u m n . T y p e 11 H R P is a less active, less highly purified form than T y p e VI, a n d these differences m a y be due to variation in the effective c o n c e n t r a t i o n o f H R P at the site o f injection.
1 AKERT, K., AND ANDY, O. J., Experimental studies on corpus mammillare and tegmento-mammillary system in the cat, Amer. J. Physiol., 183 (1955) 591. 2 CAJAL, S. RAMON Y, Histologie du SystOme Nerveux de L'Homme et des Vert~brds, Vol. 11,
Maloine, Paris, 1911, pp. 456-474. 3 COWAN, W. M., GUILLERY, R. W., AND POWELL, T. P. S., The origin of the mammillary peduncle and other hypothalamic connexions from the midbrain, J. Anat. (Lond.), 98 0964) 345-363. 4 DA1TZ, H. M., AND POWELL, T. P. S., Studies of the connexions of the fornix system, J. Neurol. Psychiat., 17 (1954) 75-82.
5 FRY, W. J., Quantitative delineation of the efferent anatomy of the medial mammillary nucleus of the cat, J. comp. NeuroL, 139 (1970) 321-336. 6 GRAHAM, R. C., JR., AND KARNOVSKY, M. J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. 7 GUILLERY, R. W., Degeneration in the post-commissural fornix and the mamillary peduncle of the rat, J. Anat. (Lond.), 90 (1956) 350-370. 8 KRISHNAN, N., AND SINGER, M., Penetration of peroxidase into peripheral nerve fibers, Amer. J. Anat., 136 (1973) 1-14. 9 KRISTENSSON,K., OLSSON, Y., AND SJ6STRAND, J., Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve, Brain Research, 32 (1971) 399-406. 10 LAVAIL, J. H., WINSTON, K. R., AND TISH, A., A method based on retrograde intraaxonal transport of protein for identification of cell bodies of origin of axons terminating within the CNS, Brain Research, 58 (1973) 470-477. 11 LYNCH,G., GALL, C., MENSAH,P., AND COTMAN,C. W., Horseradish peroxidase histochemistry: a new method for tracing efferent projections in the central nervous system, Brain Research, 65 (1974) 373-380. 12 NAUTA, H. J. W., PRITZ, M. B., AND LASEK, R. J., Afferents to the rat caudoputamen studied with horseradish peroxidase. An evaluation of a retrograde neuroanatomical research method, Brain Research, 67 (1974) 219-238.
