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Cell bodies of origin of serotonin-immunoreactive afferents to the inferior olivary complex of the rat
BrainResearch, 399(1986)369-373 Elsevier
369
BRE 21925
Cell bodies of origin of serotonin-immunoreactive afferents to the inferior olivary complex of the rat Georgia A. Bishop and Raymond H. Ho Department of Anatomy and Neuroscience Research Laboratory, The Ohio State University, Columbus, OH 43210 ( U.S.A. )
(Accepted 12 August 1986) Key words: Inferior olive; Serotonin; Double label: Immunohistochemistry
Previous studies have used immunohistochemistry to localize serotonin to distinct olivary nuclei in several mammalian species. However, the location of the cell bodies of origin for the serotoninergic projection to the inferior olive in any of these species was unknown. In the present study, a paradigm which combines transport of horseradish peroxidase (HRP) and serotonin immunohistochemistry (PAP) was used to identify the cell bodies of origin of this afferent system to the inferior olivary complex of the rat. Cells which contain both retrogradely transported HRP and brown cytoplasmic staining indicating that they are serotoninergic cells that project to the inferior olivary complex are found exclusively in an area dorsal to the rostrolateral dorsal accessory olive within the nucleus reticularis paragigantocellularis. Neurons within this nucleus were also found to be a source of serotoninergic afferents to the cerebellum and spinal cord of the rat. This raises the possibilitythat individual serotonin-immunoreactive neurons within this nucleus may project to all 3 areas. Future studies will be designed to address this possibility. No double-labeled cells were observed within any of the raphe nuclei, Several studies 1,9,14A7,19,21 have described serotoninimmunoreactive fibers and varicosities within distinct nuclei of the m a m m a l i a n inferior olivary complex. Based on physiological studies, it has been p r o p o s e d that serotoninergic afferents to the inferior olive are involved in producing harmaline-induced tremors 14. It is suggested 14 that serotonin exerts a tonic inhibitory effect on olivary neurons and that harmaline prevents this action. The olivary neurons that are thus released from this inhibitory effect fire at an increased frequency which results in a visible tremor. Although this m o n o a m i n e has been localized within the inferior olive, the location of the cell bodies of origin for this afferent system is unknown. Therefore, in the present study, a double-labeling paradigm which combines r e t r o g r a d e transport of horseradish peroxidase ( H R P ) with immunohistochemistry has been used to identify the cell bodies of origin for the serotoninergic projection to the inferior olive of the rat. Seven h o o d e d rats (225-300 g) were anesthetized with a combination of k e t a m i n e (9 mg/100 g) and xy-
lazine (1 mg/100 g) given intramuscularly. The surgical approach, including the p r o c e d u r e s for injecting small amounts of H R P into the inferior olive are described in a previous r e p o r t 2. Following survivals of 24-48 h, the animals were deeply anesthetized with sodium p e n t o b a r b i t a l (6 mg/100 g) and perfused through the ascending aorta with saline followed by 3.5% p a r a f o r m a l d e h y d e in phosphate buffer (pH 7.4). The tissue was processed for H R P histochemistry followed by serotonin immunohistochemistry as described in a previous p a p e r 3. The primary antibody (194D) was obtained from Dr. R o b e r t E l d e from the University of Minnesota. This antibody has been characterized and was used in a previous study which described the distribution of serotonin immunoreactivity in the inferior olivary complex of the rat 1. Figs. 1 - 3 are c a m e r a lucida drawings of brainstem sections from 3 cases, in which the H R P injections were placed in medial (Fig. 1), intermediate (Fig. 2) or lateral (Fig. 3) regions of the nuclear complex. The drawn sections in each figure are s e p a r a t e d by approximately 180 ftm and the labeled cells from 3
Correspondence: G.A. Bishop, Department of Anatomy, The Ohio State University, 1645 Nell Ave., Columbus, OH 43210, U.S.A.
37O
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® Figs. 1-3. Camera lucida drawings of representative transverse sections of the rat medulla; adjacent sections are separated by a distance of approximately 180/~m. The dense stippling indicates the site of the HRP injection in the inferior olivary complex. The light stippling indicates diffuse spread of the HRP reaction product. The closed circles on each drawing represent serotoninergic neurons, the stars represent cells that are retrogradely labeled with HRP and the asterisks indicate serotoninergic cells that also contain retrogradely transported HRP. Cells from 3 adjacent sections are plotted on each section illustrated. Each symbol represents one cell. DAO, dorsal accessory olive; Gi, n. reticularis gigantocellularis: MAO, medial accessory olive: MVe, medial vestibular nucleus; PGi, n. reticularis paragigantocellularis; PO, principal olivary nucleus; PrH, perihypoglossal nucleus: RM, n. raphe magnus: RO, n. raphe obscurus; Sol, n. solitarius; V, trigeminal nucleus; XIL hypoglossal nucleus.
371
® adjacent serial sections have been plotted on each of the illustrated sections. In each case illustrated, as well as those not shown, the injection of H R P was confined to the olivary complex. There was no spread of the injection beyond either the caudal or rostral poles of the nucleus. The stippling within the pyramids in Fig. 1 at levels rostral to the olive represents injury filled fibers located within the pyramids. Three types of labeled cells are observed: (1) cells that contain only retrogradely transported H R P which are characterized by the presence of a black granular reaction product within their cytoplasm (Figs. 1-3, stars, 5; (2) cells which show only brown immunostaining in their cytoplasm which indicates serotonin immunoreactivity (Figs. 1-3, closed circles, 4) and (3) cells which contain both the retrogradely transported H R P as well as brown immunostaining within their cytoplasm, indicating that they are serotonin-immunoreactive cells that project to the inferior olive (Figs. 1-3, asterisks, 6, 7). Retrogradely labeled cells (groups 1 and 3 above) are lo-
cated in all reticular and raphe nuclei previously described as projecting to the inferior olive 2'2°, including the nucleus raphe obscurus, nucleus reticularis paragigantocellularis, and the nucleus reticularis gigantocellularis. A few injury-filled cells are present in the hypoglossal nucleus in cases in which the twelfth cranial nerve was included in the injection (Fig. 2). The retrograde labeling in these cells is characterized by a diffuse black staining of their cytoplasm as opposed to the granular reaction product seen in cells that are not injury filled. Serotonin-immunoreactive neurons (group 2 above) are located within midline nuclei including the nucleus (n.) raphe obscurus, n. raphe pallidus and n. raphe magnus. In addition, serotoninergic neurons are also present just dorsal to the rostrolateral dorsal accessory olive (Figs. 1-3, 4). This region corresponds to the n. reticularis paragigantocellularis described by Paxinos and Watson 12. Rostral to the olivary complex, the number of serotonin-immunoreactive neurons increases as they extend medially and merge with the seroto-
372
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Figs. 4-7. Photomicrographs of single and double labeled reticular neurons. Fig. 4 is a low power micrograph of the rat's brainstem at the level of the rostral inferior olivary complex (IOC). Serotoninergic cells are present in raphe nuclei (open block arrow 1) and just dorsal to the lateral dorsal accessory olive (open block arrow 2) in the n. reticularis paragigantocellularis. The cell indicated by the small arrow is shown at higher magnification in Fig. 6. Calibration bar = 500 pm. Fig. 5: neuron in the n. reticularis paragigantocellularis that is only retrogradely labeled with HRP (closed block arrow). Figs. 6 and 7: neurons in the n. reticularis paragigantocellularis that are both retrogradely labeled with HRP and immunolabeled for serotonin. Note diffuse staining in their cytoplasm. Compare this to the lack of a diffuse cytoplasmic staining in the retrogradely labeled cell, Fig. 5. The small arrows indicate granular HRP reaction product. The calibration bar in Fig. 5 = 25 pm and also applies to Figs. 6 and 7.
nin-positive cells located in the n. r a p h e m a g n u s (Figs. 1 - 3 ) . T h e location of the s e r o t o n i n e r g i c n e u -
those cases in which the i n j e c t i o n is located m e d i a l l y (Fig. 1); these latter i n j e c t i o n s r e t r o g r a d e l y label
rons o b s e r v e d in the p r e s e n t study agrees with the findings of other investigators 5'6'7~8'16-18. Serotoniner-
m o r e cells in the raphe t h a n do i n j e c t i o n s located m o r e laterally (Figs. 2 a n d 3). These data suggest
gic cells that also c o n t a i n r e t r o g r a d e l y t r a n s p o r t e d
that the s e r o t o n i n e r g i c p r o j e c t i o n to the inferior olive m a y be restricted to a single n u c l e u s within the reticular f o r m a t i o n of the rat, the n. reticularis paragigantocellularis.
H R P , which indicates that they p r o j e c t to the i n f e r i o r olive, are located exclusively in the n. reticularis paragigantocellularis. N o such d o u b l e - l a b e l e d cells are o b s e r v e d w i t h i n a n y of the r a p h e nuclei e v e n in
N e u r o n s w i t h i n the n. reticularis p a r a g i g a n t o c e l l u -
373 laris were found to be a source of serotoninergic afferents to the rat's cerebellum 3. In separate studies 4'15, the double labeling technique revealed that a
dress this question. In summary, the present study has used a double label paradigm to identify the cells that give rise to se-
labeled cells identified in the present study also give rise to a monoaminergic input to the cerebellum and/
rotoninergic afferents to the inferior olive. These cells are located within the immediate vicinity of the olivary complex in the n. reticularis paragigantocellularis. A few double-labeled cells are also seen in this same nucleus at levels just rostral to the inferior
or spinal cord. However, in the present study, cells
olive. The raphe nuclei do not appear to participate
that only contain the H R P reaction product intermingle with double-labeled cells. Thus, it is possible
in the serotoninergic innervation of the inferior olive.
that neurons in the n. reticularis paragigantocellula-
This work was supported by N I H G r a n t NS 18028. The authors are grateful for the technical assistance
portion of the serotoninergic projection to the spinal cord was derived from n e u r o n s located within this nucleus. Therefore, it is possible that the double-
ris, which contain a different putative neurotransmitter, project to the inferior olive, cerebellum3 and the
of Mrs. Katharine Dillingham and the photographic
spinal cord a°'la'a3. F u t u r e studies are needed to ad-
assistance of Mr. Karl Rubin.
1 Bishop, G.A. and Ho, R.H., Substance P and serotonin immunoreactivity in the rat inferior olive, Brain Res. Bull., 12 (1984) 105-113. 2 Bishop, G.A., The origin of the reticulo-olivary projection in the rat: a retrograde horseradish peroxidase study, Neuroscience, 11 (1984) 487-496. 3 Bishop, G.A. and Ho, R.H., The distribution and origin of serotonin immunoreactivity in the rat cerebellum, Brain Research, 331 (1985) 195-207. 4 Bowker, R.M., Steinbusch, H.W.M. and Coulter, J.D., Serotonergic and peptidergic projections to the spinal cord demonstrated by a combined retrograde HRP histochemical and immunocytochemical staining method, Brain Research, 211 (1981) 412-417. 5 Butcher, L.L. and Woolf, N.J., Cholinergic and serotonergic systems in the brain and spinal cord: anatomic organization, role in intercellular communication processes, and interactive mechanisms, Prog, Brain Res., 55 (1982) 1-40. 6 Dahlstrom, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system, Acta Physiol. Scand., 62 (1964) 2-55. 7 Howe, P.R.C., Moon, E. and Dampney, R.A.L., Distribution of serotonin nerve cells in the rabbit brainstem, Neurosci. Lett., 38 (1983) 125-130. 8 Hunt, S.P. and Lovick, T.A., The distribution of serotonin, Met-enkephalin and fl-lipotropin-like immunoreactivity in neuronal perikarya of the cat brainstem, Neurosci. Lett., 30 (1982) 139-145. 9 King, J.S., Ho, R.H. and Burry, R.W., The distribution and synaptic organization of serotoninergic elements in the inferior olivary complex of the opossum, J. Comp. Neurol., 227 (1984) 357-368. 10 Leichnetz, G.R., Watkins, L., Griffin, G., Murfin, R. and Mayer, D.J., The projection from the nucleus raphe magnus and other brainstem nuclei to the spinal cord in the rat: a study using the HRP blue-reaction, Neurosci. Lett., 8 (1978) 119-124.
11 Martin, G.F., Vertes, R.P. and Waltzer, R., Spinal projections of the gigantocellular reticular formation in the rat. Evidence for projections from different areas to laminae I and II and lamina IX, Exp. Brain Res., 58 (1985) 154-162. 12 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates. Academic Press, Australia, 1982. 13 Satoh, K., The origin of reticulospinal fibers in the rat: an HRP study, J. Hirnforsch., 20 (1979) 313-332. 14 Sjolund, B., Bjorklund, A. and Wiklund, L., The indolaminergic innervation of the inferior olive. 2. Relation to hatmaline-induced tremor, Brain Research, 131 (1977) 23-37. 15 Skagerberg, G. and Bjorklund, A., topographic principles in the spinal projections of serotonergic and non-serotonergic brainstem neurons in the rat, Neuroscience, 15 (1985) 445-480. 16 Sladek, J.R. and Hoffman, G.E., Monoaminergic innervation of the mammalian inferior olivary complex. In J. Courville et al. (Eds.), The Inferior Olivary Nucleus: Anatomy and Physiology. Raven Press, New York, 1980, pp. 145-162.
17 Steinbusch, H.W.M., Distribution of serotonin-immunoreactivity in the central nervous system of the rat - - cell bodies and terminals, Neuroscience, 6 (1981) 557-618. 18 Takeuchi, Y., Kimura, H. and Sano, Y., Immunohistochemical demonstration of the distribution of serotonin neurons in the brainstem of the rat and cat, Cell Tissue Res., 24 (1982) 247-267. 19 Takeuchi, Y. and Sano, Y., Immunohistochemical demonstration of serotonin-containing nerve fibers in the inferior olivary complex of the rat, cat and monkey, Cell Tissue Res., 231 (1983) 17-28. 20 Walberg, F. and Dietrichs, E., Olivary afferents from the raphe nuclei as studied with retrograde transport of horseradish peroxidase, Anat. Embryol., 164 (1982) 85-93. 21 Wiklund, L., Sjolund, B. and Bjorklund, A., Morphological and functional studies on the serotoninergic innervation of the inferior olive, J. Physiol. (Paris), 77 (1981) 183-186.
369
BRE 21925
Cell bodies of origin of serotonin-immunoreactive afferents to the inferior olivary complex of the rat Georgia A. Bishop and Raymond H. Ho Department of Anatomy and Neuroscience Research Laboratory, The Ohio State University, Columbus, OH 43210 ( U.S.A. )
(Accepted 12 August 1986) Key words: Inferior olive; Serotonin; Double label: Immunohistochemistry
Previous studies have used immunohistochemistry to localize serotonin to distinct olivary nuclei in several mammalian species. However, the location of the cell bodies of origin for the serotoninergic projection to the inferior olive in any of these species was unknown. In the present study, a paradigm which combines transport of horseradish peroxidase (HRP) and serotonin immunohistochemistry (PAP) was used to identify the cell bodies of origin of this afferent system to the inferior olivary complex of the rat. Cells which contain both retrogradely transported HRP and brown cytoplasmic staining indicating that they are serotoninergic cells that project to the inferior olivary complex are found exclusively in an area dorsal to the rostrolateral dorsal accessory olive within the nucleus reticularis paragigantocellularis. Neurons within this nucleus were also found to be a source of serotoninergic afferents to the cerebellum and spinal cord of the rat. This raises the possibilitythat individual serotonin-immunoreactive neurons within this nucleus may project to all 3 areas. Future studies will be designed to address this possibility. No double-labeled cells were observed within any of the raphe nuclei, Several studies 1,9,14A7,19,21 have described serotoninimmunoreactive fibers and varicosities within distinct nuclei of the m a m m a l i a n inferior olivary complex. Based on physiological studies, it has been p r o p o s e d that serotoninergic afferents to the inferior olive are involved in producing harmaline-induced tremors 14. It is suggested 14 that serotonin exerts a tonic inhibitory effect on olivary neurons and that harmaline prevents this action. The olivary neurons that are thus released from this inhibitory effect fire at an increased frequency which results in a visible tremor. Although this m o n o a m i n e has been localized within the inferior olive, the location of the cell bodies of origin for this afferent system is unknown. Therefore, in the present study, a double-labeling paradigm which combines r e t r o g r a d e transport of horseradish peroxidase ( H R P ) with immunohistochemistry has been used to identify the cell bodies of origin for the serotoninergic projection to the inferior olive of the rat. Seven h o o d e d rats (225-300 g) were anesthetized with a combination of k e t a m i n e (9 mg/100 g) and xy-
lazine (1 mg/100 g) given intramuscularly. The surgical approach, including the p r o c e d u r e s for injecting small amounts of H R P into the inferior olive are described in a previous r e p o r t 2. Following survivals of 24-48 h, the animals were deeply anesthetized with sodium p e n t o b a r b i t a l (6 mg/100 g) and perfused through the ascending aorta with saline followed by 3.5% p a r a f o r m a l d e h y d e in phosphate buffer (pH 7.4). The tissue was processed for H R P histochemistry followed by serotonin immunohistochemistry as described in a previous p a p e r 3. The primary antibody (194D) was obtained from Dr. R o b e r t E l d e from the University of Minnesota. This antibody has been characterized and was used in a previous study which described the distribution of serotonin immunoreactivity in the inferior olivary complex of the rat 1. Figs. 1 - 3 are c a m e r a lucida drawings of brainstem sections from 3 cases, in which the H R P injections were placed in medial (Fig. 1), intermediate (Fig. 2) or lateral (Fig. 3) regions of the nuclear complex. The drawn sections in each figure are s e p a r a t e d by approximately 180 ftm and the labeled cells from 3
Correspondence: G.A. Bishop, Department of Anatomy, The Ohio State University, 1645 Nell Ave., Columbus, OH 43210, U.S.A.
37O
CAUDAL
Q
~'T ~
RAL
® Figs. 1-3. Camera lucida drawings of representative transverse sections of the rat medulla; adjacent sections are separated by a distance of approximately 180/~m. The dense stippling indicates the site of the HRP injection in the inferior olivary complex. The light stippling indicates diffuse spread of the HRP reaction product. The closed circles on each drawing represent serotoninergic neurons, the stars represent cells that are retrogradely labeled with HRP and the asterisks indicate serotoninergic cells that also contain retrogradely transported HRP. Cells from 3 adjacent sections are plotted on each section illustrated. Each symbol represents one cell. DAO, dorsal accessory olive; Gi, n. reticularis gigantocellularis: MAO, medial accessory olive: MVe, medial vestibular nucleus; PGi, n. reticularis paragigantocellularis; PO, principal olivary nucleus; PrH, perihypoglossal nucleus: RM, n. raphe magnus: RO, n. raphe obscurus; Sol, n. solitarius; V, trigeminal nucleus; XIL hypoglossal nucleus.
371
® adjacent serial sections have been plotted on each of the illustrated sections. In each case illustrated, as well as those not shown, the injection of H R P was confined to the olivary complex. There was no spread of the injection beyond either the caudal or rostral poles of the nucleus. The stippling within the pyramids in Fig. 1 at levels rostral to the olive represents injury filled fibers located within the pyramids. Three types of labeled cells are observed: (1) cells that contain only retrogradely transported H R P which are characterized by the presence of a black granular reaction product within their cytoplasm (Figs. 1-3, stars, 5; (2) cells which show only brown immunostaining in their cytoplasm which indicates serotonin immunoreactivity (Figs. 1-3, closed circles, 4) and (3) cells which contain both the retrogradely transported H R P as well as brown immunostaining within their cytoplasm, indicating that they are serotonin-immunoreactive cells that project to the inferior olive (Figs. 1-3, asterisks, 6, 7). Retrogradely labeled cells (groups 1 and 3 above) are lo-
cated in all reticular and raphe nuclei previously described as projecting to the inferior olive 2'2°, including the nucleus raphe obscurus, nucleus reticularis paragigantocellularis, and the nucleus reticularis gigantocellularis. A few injury-filled cells are present in the hypoglossal nucleus in cases in which the twelfth cranial nerve was included in the injection (Fig. 2). The retrograde labeling in these cells is characterized by a diffuse black staining of their cytoplasm as opposed to the granular reaction product seen in cells that are not injury filled. Serotonin-immunoreactive neurons (group 2 above) are located within midline nuclei including the nucleus (n.) raphe obscurus, n. raphe pallidus and n. raphe magnus. In addition, serotoninergic neurons are also present just dorsal to the rostrolateral dorsal accessory olive (Figs. 1-3, 4). This region corresponds to the n. reticularis paragigantocellularis described by Paxinos and Watson 12. Rostral to the olivary complex, the number of serotonin-immunoreactive neurons increases as they extend medially and merge with the seroto-
372
....
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2 :
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Figs. 4-7. Photomicrographs of single and double labeled reticular neurons. Fig. 4 is a low power micrograph of the rat's brainstem at the level of the rostral inferior olivary complex (IOC). Serotoninergic cells are present in raphe nuclei (open block arrow 1) and just dorsal to the lateral dorsal accessory olive (open block arrow 2) in the n. reticularis paragigantocellularis. The cell indicated by the small arrow is shown at higher magnification in Fig. 6. Calibration bar = 500 pm. Fig. 5: neuron in the n. reticularis paragigantocellularis that is only retrogradely labeled with HRP (closed block arrow). Figs. 6 and 7: neurons in the n. reticularis paragigantocellularis that are both retrogradely labeled with HRP and immunolabeled for serotonin. Note diffuse staining in their cytoplasm. Compare this to the lack of a diffuse cytoplasmic staining in the retrogradely labeled cell, Fig. 5. The small arrows indicate granular HRP reaction product. The calibration bar in Fig. 5 = 25 pm and also applies to Figs. 6 and 7.
nin-positive cells located in the n. r a p h e m a g n u s (Figs. 1 - 3 ) . T h e location of the s e r o t o n i n e r g i c n e u -
those cases in which the i n j e c t i o n is located m e d i a l l y (Fig. 1); these latter i n j e c t i o n s r e t r o g r a d e l y label
rons o b s e r v e d in the p r e s e n t study agrees with the findings of other investigators 5'6'7~8'16-18. Serotoniner-
m o r e cells in the raphe t h a n do i n j e c t i o n s located m o r e laterally (Figs. 2 a n d 3). These data suggest
gic cells that also c o n t a i n r e t r o g r a d e l y t r a n s p o r t e d
that the s e r o t o n i n e r g i c p r o j e c t i o n to the inferior olive m a y be restricted to a single n u c l e u s within the reticular f o r m a t i o n of the rat, the n. reticularis paragigantocellularis.
H R P , which indicates that they p r o j e c t to the i n f e r i o r olive, are located exclusively in the n. reticularis paragigantocellularis. N o such d o u b l e - l a b e l e d cells are o b s e r v e d w i t h i n a n y of the r a p h e nuclei e v e n in
N e u r o n s w i t h i n the n. reticularis p a r a g i g a n t o c e l l u -
373 laris were found to be a source of serotoninergic afferents to the rat's cerebellum 3. In separate studies 4'15, the double labeling technique revealed that a
dress this question. In summary, the present study has used a double label paradigm to identify the cells that give rise to se-
labeled cells identified in the present study also give rise to a monoaminergic input to the cerebellum and/
rotoninergic afferents to the inferior olive. These cells are located within the immediate vicinity of the olivary complex in the n. reticularis paragigantocellularis. A few double-labeled cells are also seen in this same nucleus at levels just rostral to the inferior
or spinal cord. However, in the present study, cells
olive. The raphe nuclei do not appear to participate
that only contain the H R P reaction product intermingle with double-labeled cells. Thus, it is possible
in the serotoninergic innervation of the inferior olive.
that neurons in the n. reticularis paragigantocellula-
This work was supported by N I H G r a n t NS 18028. The authors are grateful for the technical assistance
portion of the serotoninergic projection to the spinal cord was derived from n e u r o n s located within this nucleus. Therefore, it is possible that the double-
ris, which contain a different putative neurotransmitter, project to the inferior olive, cerebellum3 and the
of Mrs. Katharine Dillingham and the photographic
spinal cord a°'la'a3. F u t u r e studies are needed to ad-
assistance of Mr. Karl Rubin.
1 Bishop, G.A. and Ho, R.H., Substance P and serotonin immunoreactivity in the rat inferior olive, Brain Res. Bull., 12 (1984) 105-113. 2 Bishop, G.A., The origin of the reticulo-olivary projection in the rat: a retrograde horseradish peroxidase study, Neuroscience, 11 (1984) 487-496. 3 Bishop, G.A. and Ho, R.H., The distribution and origin of serotonin immunoreactivity in the rat cerebellum, Brain Research, 331 (1985) 195-207. 4 Bowker, R.M., Steinbusch, H.W.M. and Coulter, J.D., Serotonergic and peptidergic projections to the spinal cord demonstrated by a combined retrograde HRP histochemical and immunocytochemical staining method, Brain Research, 211 (1981) 412-417. 5 Butcher, L.L. and Woolf, N.J., Cholinergic and serotonergic systems in the brain and spinal cord: anatomic organization, role in intercellular communication processes, and interactive mechanisms, Prog, Brain Res., 55 (1982) 1-40. 6 Dahlstrom, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system, Acta Physiol. Scand., 62 (1964) 2-55. 7 Howe, P.R.C., Moon, E. and Dampney, R.A.L., Distribution of serotonin nerve cells in the rabbit brainstem, Neurosci. Lett., 38 (1983) 125-130. 8 Hunt, S.P. and Lovick, T.A., The distribution of serotonin, Met-enkephalin and fl-lipotropin-like immunoreactivity in neuronal perikarya of the cat brainstem, Neurosci. Lett., 30 (1982) 139-145. 9 King, J.S., Ho, R.H. and Burry, R.W., The distribution and synaptic organization of serotoninergic elements in the inferior olivary complex of the opossum, J. Comp. Neurol., 227 (1984) 357-368. 10 Leichnetz, G.R., Watkins, L., Griffin, G., Murfin, R. and Mayer, D.J., The projection from the nucleus raphe magnus and other brainstem nuclei to the spinal cord in the rat: a study using the HRP blue-reaction, Neurosci. Lett., 8 (1978) 119-124.
11 Martin, G.F., Vertes, R.P. and Waltzer, R., Spinal projections of the gigantocellular reticular formation in the rat. Evidence for projections from different areas to laminae I and II and lamina IX, Exp. Brain Res., 58 (1985) 154-162. 12 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates. Academic Press, Australia, 1982. 13 Satoh, K., The origin of reticulospinal fibers in the rat: an HRP study, J. Hirnforsch., 20 (1979) 313-332. 14 Sjolund, B., Bjorklund, A. and Wiklund, L., The indolaminergic innervation of the inferior olive. 2. Relation to hatmaline-induced tremor, Brain Research, 131 (1977) 23-37. 15 Skagerberg, G. and Bjorklund, A., topographic principles in the spinal projections of serotonergic and non-serotonergic brainstem neurons in the rat, Neuroscience, 15 (1985) 445-480. 16 Sladek, J.R. and Hoffman, G.E., Monoaminergic innervation of the mammalian inferior olivary complex. In J. Courville et al. (Eds.), The Inferior Olivary Nucleus: Anatomy and Physiology. Raven Press, New York, 1980, pp. 145-162.
17 Steinbusch, H.W.M., Distribution of serotonin-immunoreactivity in the central nervous system of the rat - - cell bodies and terminals, Neuroscience, 6 (1981) 557-618. 18 Takeuchi, Y., Kimura, H. and Sano, Y., Immunohistochemical demonstration of the distribution of serotonin neurons in the brainstem of the rat and cat, Cell Tissue Res., 24 (1982) 247-267. 19 Takeuchi, Y. and Sano, Y., Immunohistochemical demonstration of serotonin-containing nerve fibers in the inferior olivary complex of the rat, cat and monkey, Cell Tissue Res., 231 (1983) 17-28. 20 Walberg, F. and Dietrichs, E., Olivary afferents from the raphe nuclei as studied with retrograde transport of horseradish peroxidase, Anat. Embryol., 164 (1982) 85-93. 21 Wiklund, L., Sjolund, B. and Bjorklund, A., Morphological and functional studies on the serotoninergic innervation of the inferior olive, J. Physiol. (Paris), 77 (1981) 183-186.