Brain Research, 108 (1976) 165-169
165
© ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands
Direct projections from hypothalamus to hippocampus in the rat demonstrated by retrograde transport of horseradish peroxidase
DANIEL A. PASQUIER* AND FERNANDO REINOSO-SUAREZ Departamento de Morfologia, Facuhad de Medicina, Universidad Aut6noma de Madrid, Madrid 34 (Spain)
(Accepted February 10th, 1976)
The hypothalamus and hippocampus have been correlated by both functional and morphological approachesS, 11. Direct hypothalamic projections to the hippocampus have been reported from studies with silver impregnation methods2, but it now seems possible that some of these connecting fibers may be by-passing fibers from caudal regionsT,a. Recently, in cases of hippocampal injection of horseradish peroxidase (HRP), labeled neurons were found in the supramammillary region9, an observation suggesting the existence of direct hypothalamo-hippocampal projections. In the present study the distribution of hypothalamic neurons labeled by HRP injections in the hippocampus was investigated in some more detail, and an attempt was made to establish topographical correlations between the two brain regions. Stereotaxic injections of 0.1-0.2 /zl of 50~ HRP solution (Sigma type VI) were made into the right or left hippocampus of 24 white rats (150-250 g) trying to avoid diffusion of HRP to neighboring regions. Sixteen rats were injected at various rostrocaudal points in the dorsal hippocampus. In 3 animals the injection was localized to the ventral hippocampus (the needle was introduced through the cortex in an oblique direction to avoid the dorsal hippocampus). In 5 control rats HRP was injected at different sites in the cortex overlying the hippocampus. After a 24-h survival time, the animals were perfused, and the brains sectioned on a freezing microtome in the frontal, horizontal or sagittal plane. The sections were processed according to the method of Llamas and Martinez-Morenoa. Labeled neurons were not found in any hypothalamic nuclei when HRP had been injected in any of the following places: the cortex above and lateral to the hippocampus, the anterior portion of the dorsal hippocampus, the subiculum of the dorsal posterior hippocampus (Fig. 1) and the anterior portion of the ventral hippocampus (not represented in the figure). Since HRP-filled neurons were observed in all these cases in other brain regions, it seems unlikely that this negative result is attributable to technical failure. * Present address: The WorcesterFoundationfor ExperimentalBiology,Shrewsbury, Mass. 01545, U.S.A.
166
Fig. 1. HRP injections placed at the dorsal anterior (A) and posterior (B) hippocampus showing: no labeled HRP neurons in the hypothalamus (open areas in thin line); abundant HRP neurons appear in the submammillothalamic and supramammillary hypothalamic nuclei (dotted areas in thick line); scarce HRP neurons only in the ipsilateral supramammillary nucleus (dotted areas in thin line). Fi, fimbria. L a b e l e d n e u r o n s were observed in the h y p o t h a l a m u s in cases o f H R P injection in the d o r s a l p o s t e r i o r h i p p o c a m p u s (Fig. 1); each injection always i n v o l v e d CA1 a n d C A 4 a n d in some cases also the gyrus d e n t a t u s a n d CA3. In all these a n i m a l s H R P - p o s i t i v e n e u r o n s a p p e a r e d in the s u p r a m a m m i l l a r y nucleus a n d in a n o t h e r cell g r o u p l o c a t e d below the m a m m i l l o t h a l a m i c bundle (Figs. 1-3).
Fig. 2. Diagrams of frontal (A) and horizontal (B) sections of two rat brains with HRP injections into the dorsal posterior hippocampus (A3, B2). Dots represent HRP neurons. CS, colliculus superior; F, fornix; Ip, nucleus interpeduncularis; LM, lemniscus medialis; M, nucleus mammillaris, MFB, medial forebrain bundle; Mtb, L mammillothalamicus; Sm, nucleus supramammillaris; Smt, nucleus submammiUothalamicus; Sth, nucleus subthalamicus; TO, t. opticus.
167
Fig. 3. A: dark-field photomicrograph of HRP neurons of the supramammillary nucleus ipsilateral to the injection into the dorsal posterior hippocampus ( x 150). B: labeled neurons in the supramammillary nucleus ( x 500). C: coronal section at the level of the posterior hypothalamus showing the localization (arrows) of the submammillothalamic nucleus. D: dark field photomicrograph of a horizontal section showing HRP neurons of the submammillothalamic nucleus ( x 150). E: labeled neurons in the submammillothalamic nucleus ( x 500).
The s u p r a m a m m i l l a r y nucleus (Sm) is situated d o r s a l to b o t h the lateral a n d m e d i a l m a m m i l l a r y nuclei. A f t e r H R P injection in the d o r s a l p o s t e r i o r h i p p o c a m p u s , n u m e r o u s l a b e l e d n e u r o n s a p p e a r e d a l m o s t t h r o u g h o u t the ipsilateral Sm (Figs.
168 2 A a n d 3A); only a few HRP neurons appeared in the contralateral Sm. After HRP injection in the most posterior portion of the dorsal hippocampus a small number of labeled neurons were observed only in the ipsilateral Sm. The cell region located below the mammillothalamic bundle (submammillothalamic nucleus, Smt) in which numerous additional labeled neurons were found after HRP injection in the dorsal posterior hippocampus, corresponds to the lateral border of the dorsal and dorsocaudal hypothalamic area of Diepen 1 and to the dorsal and posterior hypothalamic nuclei of other authors 5. The Smt is placed medial to the zona incerta and the medial forebrain bundle, MFB (Fig. 3C); in sagittal sections it appears in the angle between fornix and mammillothalamic tract; in the frontal plane it shows a cup-like shape and, accordingly, in horizontal sections the cells appear to surround the mammillothalamic tract (Figs. 2B and 3D). The majority of these cells are of medium size, some are larger, and the latter are comparable to those of the zona incerta and lateral hypothalamus. The appearance of Smt is different from the more ventral, easily identifiable perifornical nucleus. The dorsal border of this neuronal group is a bed nucleus of the mammillothalamic tract but its neurons can be distinguished from the cells in a parafascicular location. After HRP injection in the ventral hippocampus without contamination of the dorsal hippocampus, the only cells to appear labeled in the hypothalamus were sporadic neurons in the ipsilateral supramammillary nucleus. These findings demonstrate a direct connection between the hypothalamus and hippocampus, not only from the supramammillary area as described 9 but also from the submammillothatamic nucleus, situated at the lateral border of the dorsal and dorsocaudal hypothalamic region. Furthermore, in several cases the HRP neurons in the supramammillary region sharply identified and delimited the supramammillary nucleus; no labeled neurons appeared lateral to this nucleus. The largest number of labeled Sm neurons were found in cases of HRP injection in the dorsal posterior hippocampus, and only sporadic cells were labeled by ventral hippocampal injections. Ingeneral, the number of labeled hypothalamic neurons seemed to depend more on the localization than on the size of the injection. Some HRP neurons were also found in the contralateral Sm after dorsal posterior hippocampal injections, a finding not previously reported 9. The only hypothalamic lesion, located in the supramammillary region and also affecting the Smt, after which Guillery ~ found hippocampal degeneration, probably interrupts both the caudal brain stem projection to hippocampus 8 and the fibers originating in the Sm and Smt, described in the present paper. Hypothalamo-hippocampal projections from the Smt and Sm to the dorsal posterior hippocampus would seem to reciprocate the fornix projections described by Nauta 4 from the dorsal posterior hippocampus (his hippocampal middle third) to 'the dorsal hypothalamic area' as well as to the supramammillary region of both sides. On the other hand, the caudal part of the Smt (lateral portion of the posterior hypothalamic nucleus) and the Sm appear to form the major hypothalamic distribution area of fibers ascending from the ponto-mesencephalic isthmus by way of Schiitz's dorsal longitudinal fasciculus 6.
169 Both Sm and Smt are located along the path of serotonin fibers originating in the raphe nuclei of the brain stem 1°. These fibers enter the hypothalamus dorsal to the mammillary bodies where they traverse the Sm, and pass rostrally in the medial part of the MFB, in close relation with the Smt. Along their course they issue collaterals to these hypothalamic nuclei a (Pasquier and Reinoso-Sufirez, in preparation). In summary, the present evidence indicates that the supramammillary nucleus projects to the dorsal posterior hippocampus and more sparsely to the ventral hippocampus. The submammillothalamic nucleus appears to project, by contrast, only to the dorsal posterior hippocampus with a smaller projection to the contralateral side as well. These results indicate a direct connection between the hypothalamus and the hippocampus, closing a loop with the hippocampal region that projects to this hypothalamic area. Both supramammillary and submammillothalamic nuclei, situated at the border between the lateral and medial hypothalamus, appear as important way-stations in a neural circuit extending between the ponto-mesencephalic tegmentum and the hippocampus, and could thus be involved in a variety of behavioral processes. We wish to thank Dr. Walle J. H. Nauta for his critical reading and style revision of the manuscript, and Dr. A. Llamas for his technical help.
1 DIEPEN,R., Der Hypothalamus. In W. BARGMAN(Ed.), von MOllendorff's Hdb. d. mikr. Anat. d. Menschen, Nervensystem, Vol. 4/7, Springer, Berlin, 1962. 2 GUILLERY,R. W., Degeneration in the hypothalamic connexions of the albino rat, J. Anat. (Lond.), 91 (1957) 91-115. 3 LLAMAS,A., Y MARTINEz-MORENO,E., Modificacionesal m6todo del transporte axonal retr6grado de la horseradish peroxidase en el sistema nervioso central en el gato adulto, An. Anat., 23 (1974) 3-15. 4 NAUTA, W. J. H., An experimental study of the fornix system in the rat, J. comp. Neurol., 104 (1956) 247-271. 5 NAUTA,W. J. H., ANDHAYMAKER,W., Hypothalamic nuclei and fiber connections. In W. HAYMAKER, E. ANDERSONAND W. J. H. NAUTA(Eds.), The Hypothalamus, Thomas, Springfield, Ill., 1969, pp. 136-209. 6 NAUTA,W. J. H., ANDKUYPERS,H. G. J. M., Some ascending pathways in the brain stem reticular formation. In H. H. JASPER,L. D. PROCTOR,R. S. KNIGHTON,W. C. NOSHAYANDR. T. COSTELLO (Eds.), Reticular Formation of the Brain, Little, Brown, Boston, Mass., 1957, pp. 3-30. 7 REINOSO-SUAREZ,F., NAVA,E., MARTINEZ-MORENO,E., AND FAIREN,A., Limbic and striatal afferents from the brain stem, Anat. Rec., 175 (1973) 422. 8 REINOSO-SUAREZ,F., MARTINEZ-MORENO,E., Y NAVA, B. E., Conexiones ascendentes desde el tronco del enc6falo a formaciones del hipocampo, An. Anat., 23 (1974) 95-117. 9 SEGAL,M., ANDLANDIS,S., Afferents to the hippocampus of the rat studied with the method of retrograde transport of horseradish peroxidase, Brain Research, 78 (1974) 1-15. 10 UNGERSTEDT, O., Setereotaxic mapping of the monoamine pathways in the rat brain, Acta physiol, scand., 82, Suppl. 367 (1971) 1-48. 11 VAt,a~ERWOLF,C. H., Limbic-diencephalic mechanisms of voluntary movement, Psychol. Rev.. 78 (1971) 83-113.
165
© ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands
Direct projections from hypothalamus to hippocampus in the rat demonstrated by retrograde transport of horseradish peroxidase
DANIEL A. PASQUIER* AND FERNANDO REINOSO-SUAREZ Departamento de Morfologia, Facuhad de Medicina, Universidad Aut6noma de Madrid, Madrid 34 (Spain)
(Accepted February 10th, 1976)
The hypothalamus and hippocampus have been correlated by both functional and morphological approachesS, 11. Direct hypothalamic projections to the hippocampus have been reported from studies with silver impregnation methods2, but it now seems possible that some of these connecting fibers may be by-passing fibers from caudal regionsT,a. Recently, in cases of hippocampal injection of horseradish peroxidase (HRP), labeled neurons were found in the supramammillary region9, an observation suggesting the existence of direct hypothalamo-hippocampal projections. In the present study the distribution of hypothalamic neurons labeled by HRP injections in the hippocampus was investigated in some more detail, and an attempt was made to establish topographical correlations between the two brain regions. Stereotaxic injections of 0.1-0.2 /zl of 50~ HRP solution (Sigma type VI) were made into the right or left hippocampus of 24 white rats (150-250 g) trying to avoid diffusion of HRP to neighboring regions. Sixteen rats were injected at various rostrocaudal points in the dorsal hippocampus. In 3 animals the injection was localized to the ventral hippocampus (the needle was introduced through the cortex in an oblique direction to avoid the dorsal hippocampus). In 5 control rats HRP was injected at different sites in the cortex overlying the hippocampus. After a 24-h survival time, the animals were perfused, and the brains sectioned on a freezing microtome in the frontal, horizontal or sagittal plane. The sections were processed according to the method of Llamas and Martinez-Morenoa. Labeled neurons were not found in any hypothalamic nuclei when HRP had been injected in any of the following places: the cortex above and lateral to the hippocampus, the anterior portion of the dorsal hippocampus, the subiculum of the dorsal posterior hippocampus (Fig. 1) and the anterior portion of the ventral hippocampus (not represented in the figure). Since HRP-filled neurons were observed in all these cases in other brain regions, it seems unlikely that this negative result is attributable to technical failure. * Present address: The WorcesterFoundationfor ExperimentalBiology,Shrewsbury, Mass. 01545, U.S.A.
166
Fig. 1. HRP injections placed at the dorsal anterior (A) and posterior (B) hippocampus showing: no labeled HRP neurons in the hypothalamus (open areas in thin line); abundant HRP neurons appear in the submammillothalamic and supramammillary hypothalamic nuclei (dotted areas in thick line); scarce HRP neurons only in the ipsilateral supramammillary nucleus (dotted areas in thin line). Fi, fimbria. L a b e l e d n e u r o n s were observed in the h y p o t h a l a m u s in cases o f H R P injection in the d o r s a l p o s t e r i o r h i p p o c a m p u s (Fig. 1); each injection always i n v o l v e d CA1 a n d C A 4 a n d in some cases also the gyrus d e n t a t u s a n d CA3. In all these a n i m a l s H R P - p o s i t i v e n e u r o n s a p p e a r e d in the s u p r a m a m m i l l a r y nucleus a n d in a n o t h e r cell g r o u p l o c a t e d below the m a m m i l l o t h a l a m i c bundle (Figs. 1-3).
Fig. 2. Diagrams of frontal (A) and horizontal (B) sections of two rat brains with HRP injections into the dorsal posterior hippocampus (A3, B2). Dots represent HRP neurons. CS, colliculus superior; F, fornix; Ip, nucleus interpeduncularis; LM, lemniscus medialis; M, nucleus mammillaris, MFB, medial forebrain bundle; Mtb, L mammillothalamicus; Sm, nucleus supramammillaris; Smt, nucleus submammiUothalamicus; Sth, nucleus subthalamicus; TO, t. opticus.
167
Fig. 3. A: dark-field photomicrograph of HRP neurons of the supramammillary nucleus ipsilateral to the injection into the dorsal posterior hippocampus ( x 150). B: labeled neurons in the supramammillary nucleus ( x 500). C: coronal section at the level of the posterior hypothalamus showing the localization (arrows) of the submammillothalamic nucleus. D: dark field photomicrograph of a horizontal section showing HRP neurons of the submammillothalamic nucleus ( x 150). E: labeled neurons in the submammillothalamic nucleus ( x 500).
The s u p r a m a m m i l l a r y nucleus (Sm) is situated d o r s a l to b o t h the lateral a n d m e d i a l m a m m i l l a r y nuclei. A f t e r H R P injection in the d o r s a l p o s t e r i o r h i p p o c a m p u s , n u m e r o u s l a b e l e d n e u r o n s a p p e a r e d a l m o s t t h r o u g h o u t the ipsilateral Sm (Figs.
168 2 A a n d 3A); only a few HRP neurons appeared in the contralateral Sm. After HRP injection in the most posterior portion of the dorsal hippocampus a small number of labeled neurons were observed only in the ipsilateral Sm. The cell region located below the mammillothalamic bundle (submammillothalamic nucleus, Smt) in which numerous additional labeled neurons were found after HRP injection in the dorsal posterior hippocampus, corresponds to the lateral border of the dorsal and dorsocaudal hypothalamic area of Diepen 1 and to the dorsal and posterior hypothalamic nuclei of other authors 5. The Smt is placed medial to the zona incerta and the medial forebrain bundle, MFB (Fig. 3C); in sagittal sections it appears in the angle between fornix and mammillothalamic tract; in the frontal plane it shows a cup-like shape and, accordingly, in horizontal sections the cells appear to surround the mammillothalamic tract (Figs. 2B and 3D). The majority of these cells are of medium size, some are larger, and the latter are comparable to those of the zona incerta and lateral hypothalamus. The appearance of Smt is different from the more ventral, easily identifiable perifornical nucleus. The dorsal border of this neuronal group is a bed nucleus of the mammillothalamic tract but its neurons can be distinguished from the cells in a parafascicular location. After HRP injection in the ventral hippocampus without contamination of the dorsal hippocampus, the only cells to appear labeled in the hypothalamus were sporadic neurons in the ipsilateral supramammillary nucleus. These findings demonstrate a direct connection between the hypothalamus and hippocampus, not only from the supramammillary area as described 9 but also from the submammillothatamic nucleus, situated at the lateral border of the dorsal and dorsocaudal hypothalamic region. Furthermore, in several cases the HRP neurons in the supramammillary region sharply identified and delimited the supramammillary nucleus; no labeled neurons appeared lateral to this nucleus. The largest number of labeled Sm neurons were found in cases of HRP injection in the dorsal posterior hippocampus, and only sporadic cells were labeled by ventral hippocampal injections. Ingeneral, the number of labeled hypothalamic neurons seemed to depend more on the localization than on the size of the injection. Some HRP neurons were also found in the contralateral Sm after dorsal posterior hippocampal injections, a finding not previously reported 9. The only hypothalamic lesion, located in the supramammillary region and also affecting the Smt, after which Guillery ~ found hippocampal degeneration, probably interrupts both the caudal brain stem projection to hippocampus 8 and the fibers originating in the Sm and Smt, described in the present paper. Hypothalamo-hippocampal projections from the Smt and Sm to the dorsal posterior hippocampus would seem to reciprocate the fornix projections described by Nauta 4 from the dorsal posterior hippocampus (his hippocampal middle third) to 'the dorsal hypothalamic area' as well as to the supramammillary region of both sides. On the other hand, the caudal part of the Smt (lateral portion of the posterior hypothalamic nucleus) and the Sm appear to form the major hypothalamic distribution area of fibers ascending from the ponto-mesencephalic isthmus by way of Schiitz's dorsal longitudinal fasciculus 6.
169 Both Sm and Smt are located along the path of serotonin fibers originating in the raphe nuclei of the brain stem 1°. These fibers enter the hypothalamus dorsal to the mammillary bodies where they traverse the Sm, and pass rostrally in the medial part of the MFB, in close relation with the Smt. Along their course they issue collaterals to these hypothalamic nuclei a (Pasquier and Reinoso-Sufirez, in preparation). In summary, the present evidence indicates that the supramammillary nucleus projects to the dorsal posterior hippocampus and more sparsely to the ventral hippocampus. The submammillothalamic nucleus appears to project, by contrast, only to the dorsal posterior hippocampus with a smaller projection to the contralateral side as well. These results indicate a direct connection between the hypothalamus and the hippocampus, closing a loop with the hippocampal region that projects to this hypothalamic area. Both supramammillary and submammillothalamic nuclei, situated at the border between the lateral and medial hypothalamus, appear as important way-stations in a neural circuit extending between the ponto-mesencephalic tegmentum and the hippocampus, and could thus be involved in a variety of behavioral processes. We wish to thank Dr. Walle J. H. Nauta for his critical reading and style revision of the manuscript, and Dr. A. Llamas for his technical help.
1 DIEPEN,R., Der Hypothalamus. In W. BARGMAN(Ed.), von MOllendorff's Hdb. d. mikr. Anat. d. Menschen, Nervensystem, Vol. 4/7, Springer, Berlin, 1962. 2 GUILLERY,R. W., Degeneration in the hypothalamic connexions of the albino rat, J. Anat. (Lond.), 91 (1957) 91-115. 3 LLAMAS,A., Y MARTINEz-MORENO,E., Modificacionesal m6todo del transporte axonal retr6grado de la horseradish peroxidase en el sistema nervioso central en el gato adulto, An. Anat., 23 (1974) 3-15. 4 NAUTA, W. J. H., An experimental study of the fornix system in the rat, J. comp. Neurol., 104 (1956) 247-271. 5 NAUTA,W. J. H., ANDHAYMAKER,W., Hypothalamic nuclei and fiber connections. In W. HAYMAKER, E. ANDERSONAND W. J. H. NAUTA(Eds.), The Hypothalamus, Thomas, Springfield, Ill., 1969, pp. 136-209. 6 NAUTA,W. J. H., ANDKUYPERS,H. G. J. M., Some ascending pathways in the brain stem reticular formation. In H. H. JASPER,L. D. PROCTOR,R. S. KNIGHTON,W. C. NOSHAYANDR. T. COSTELLO (Eds.), Reticular Formation of the Brain, Little, Brown, Boston, Mass., 1957, pp. 3-30. 7 REINOSO-SUAREZ,F., NAVA,E., MARTINEZ-MORENO,E., AND FAIREN,A., Limbic and striatal afferents from the brain stem, Anat. Rec., 175 (1973) 422. 8 REINOSO-SUAREZ,F., MARTINEZ-MORENO,E., Y NAVA, B. E., Conexiones ascendentes desde el tronco del enc6falo a formaciones del hipocampo, An. Anat., 23 (1974) 95-117. 9 SEGAL,M., ANDLANDIS,S., Afferents to the hippocampus of the rat studied with the method of retrograde transport of horseradish peroxidase, Brain Research, 78 (1974) 1-15. 10 UNGERSTEDT, O., Setereotaxic mapping of the monoamine pathways in the rat brain, Acta physiol, scand., 82, Suppl. 367 (1971) 1-48. 11 VAt,a~ERWOLF,C. H., Limbic-diencephalic mechanisms of voluntary movement, Psychol. Rev.. 78 (1971) 83-113.
