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Neurokinin B peptide-2 neurons project from the hypothalamus to the thoracolumbar spinal cord of the rat
0306.4522/93 $6.00 + 0.00 Pergamon Press Ltd IBRO
Neuroscience Vol. 52. No. 4. pp. 1019-1028. 1993 Printed in Great Britain
NEUROKININ B PEPTIDE- NEURONS PROJECT FROM THE HYPOTHALAMUS TO THE THORACOLUMBAR SPINAL CORD OF THE RAT H. ZHUO and C. J. Department
of Pharmacology,
Uniformed
HELKE*
Services University MD 20814-4799. U.S.A.
of the Health
Science,
Bethesda,
Abstract-The presence and location of CNS neurokinin B peptide-2-like immunoreactive neurons that project to the spinal cord were studied by a combination of retrograde transport of fluorescent dye (FluoroGold) and fluorescence immunocytochemistry. After injections of FluoroGold into the thoracic or lumbar segments of the rat spinal cord, serial sections of brain were stained with antisera directed against neurokinin B peptide-2. The results of the study showed that neurokinin B peptide-2-like immunoreactive neurons were located in the nucleus arcuate, median eminence, ventral and external bed nuclei of the stria terminalis, dorsal hypothalamic area, and media1 habenula. Neurokinin B peptideneurons that give rise to the long descending projections from the hypothalamus to thoracolumbar spinal cord were found only in the dorsal hypothalamic area. Approximately 36% of the neurokinin B peptideneurons in the dorsal hypothalamic area projected to the spinal cord, whereas about 28% of the spinal projecting neurons in the dorsal hypothalamic area contained neurokinin B peptide-2-like immunoreactivity. Most of the spinal projecting neurokinin B peptideneurons in the dorsal hypothalamic area had a cell size of 15 x 25 pm. In the spinal cord, immunoreactive neurokinin B peptidefibers and terminals were distributed mainly in the superficial dorsal horn and the central autonomic area, with the highest density in laminae II and X, with less density in laminae IV and V. A few neurokinin peptide-? fibers and terminals were also found in the ventral horn of the spinal cord. The results of the present study show that hypothalamic neurokinin B peptideneurons are the main source of the spinal neurokinin B peptide-2.
Mammalian namely,
tachykinins substance
substance medin
K)
and
K). These
carboxyl-terminal
include
P (SP), neurokinin
tachykinin amino
a family
neurokinin
of peptides, A (NKA,
B (NKB, peptides
acid
share
sequence
or
or
neuro-
a common Phe-X-Gly-
where X is a hydrophobic or aromatic residue.8.‘b,‘8,‘9,2’Tachykinins are widely distributed in both the CNS and peripheral tissues and evoke a variety of biological activities (for review see Refs 18, 22). Four precursors of mammalian tachykinins are encoded by two distinct genes. They are c(-, b-, and y-preprotachykinin-A which contain sequences of substance P and neurokinin A,8.‘2.20 and preprotachykinin-B which contains the sequence of neurokinin B.13 Besides NKB, the preprotachykinin B contains a 30-amino acid non-tachykinin sequence (Leu-Try-Asp-Ser-Arg-Ser-Ile-Ser-Leu-Glu-GlyLeu-Leu-Lys-Val-Leu-Ser-Lys-Ala-Ser-Val-Gly-ProLys-Glu-Thr-Ser-Leu-Pro-Gin)” called peptide 2 or NKB peptide(NKB-P2). Recently, a highly specific antiserum directed against NKB-P2 was developed by Krause ef al. ” Because of the non-tachykinin structure of NKB-P2, antibodies to NKB-P2 can be Leu-Met-NH,,
*To whom correspondence should be addressed. Abbreuiafions: FG, FluoroGold; -LI, -like immunoreactive; NGS, normal goat serum; NKA, neurokinin A; NKB, neurokinin B; NKB-P2, neurokinin B peptide-2; PBS, phosphate-buffered saline; PVN. paraventricular nucleus; SP, substance P.
valuable to identify NKB neurons and fibers without the problems of cross-reaction with tachykinin peptides. Studies using in situ hybridization and immunocytochemistry on rat” and on hamster” showed that the majority of the CNS NKB-P2 neurons are located in the hypothalamus. Preprotachykinin B, the precursor for NKB and NKB-P2, was reported to have higher levels in the hypothalamus than other regions of the CNS.‘3.‘7 Preliminary experiments of the present study showed NKB-P24ike immunoreactive (-LI) fibers and terminals mainly in the superficial dorsal horn and the central autonomic area (lamina X) of the spinal cord. This distribution was distinct from that of other tachykinin (e.g. SP, NKA) fibers and terminals.8~‘0~22~28 The present study combined retrograde transport of FluoroGold (FG) and NKB-P2 immunocytochemistry to determine the site of the origin of the CNS NKB-P2 projections to the spinal cord.
EXPERIMENTAL
PROCEDURES
Animal preparation Ten male Sprague-Dawley rats (weighing 250-450 g. Taconic Farms) were used in this study. Laminectomies of thoracic or lumbar spinal cord were performed under halothane anesthesia. One to two microliters of 4% FG (Fluorochrome Inc., U.S.A.) in saline was injected 1019
unilaterally
into the thoracic
(T,,)
enlargement FG was delivered to the spinal cord via multiple injections. attempting to involve dorsal and ventral horns of the spinal cord. so as to maximally label spinally projecting neurons. After 48-72 h survival, colchicine (60 pg in 10 ~1 of saline) was administered i.c. under halothane anesthesia (2% in oxygen). Twenty-four hours later, animals were given an overdose of chloral hydrate and perfused intracardially with the following solutions: 200ml of 100 units of heparin in saline; 400 ml of ice-cold 4% paraformaldehyde in 0. I M phosphate buffer (pH 7.4). The brain and spinal cord were removed, post-fixed in the same fixative overnight, and soaked in 30% of sucrose in 0.1 M phosphate-buffered saline (PBS) for another 24 h at 4 C.
RESUI,TS
or lumbar
(L,) of the spinal cord using a glass micropipette.
Immunohistochemistq The brain was frozen, sectioned at 25 pm with a cryostat, and tissue sections were collected in PBS. Free-floating brain sections were processed with indirect immunofluorescence staining method. After pre-incubation for 30min in 3% normal goat serum (NGS) in PBS with 0.3% Triton X-100, the sections were first incubated for 48 h with the primary antiserum, rabbit NKB-P2 (generously donated by Dr Krause, Washington University), prepared in 1% NGS in 0.1 M PBS (l:lO@O), and then incubated with the fluorescein-labeled goat anti-rabbit IgG (Jackson Lab., U.S.A.) in 0.1 M PBS (1:lOO) for 1 h at room temperature. The processed sections were wet-mounted onto glass slides and coverslipped with Permafluor Aqueous Mounting Medium (Lipshaw. U.S.A.). Sections were examined and photographed with a Leitz fluorescence photomicroscope. The spinal cord segments (injection sites) were sectioned at 60 pm thickness. The injection sites for FG in the spinal cord were identified and drawn using camera lucida. Characterisrics
of the antisera
A detailed characterization of the polyclonal antisera for NKB-P2 has been described elsewhere.” Briefly, the NKB-P2 antisera were raised against a synthetic peptide corresponding to the amino acids 5@-79 of the rat preprotachykinin B.’ It does not cross-react with substance P. NKA. NKB and neuropeptide K.” Control experiments
To ascertain whether the immunocytochemical reactions were specific. three control experiments were performed on representative sections. (I) Prior to immunostaining, the diluted antiserum was absorbed with the synthetic NKB-P2 (100 pg/ml of the final diluted antiserum). (2) The primary antiserum was omitted in the first incubation. (3) Normal rabbit serum was used instead of the primary antiserum in the first incubation. The control experiments resulted in no staining in the representative tissue sections.
Abbreviations 3v AC ACe ACo Ar BST
cc
CA3 CP CPU cx DA F FR
3rd ventricle anterior commissure central nucleus of the amygdaloid complex anterior cortical nucleus of the amygdaloid complex nucleus arcuate bed nucleus of the stria terminalis central canal hippocampus cerebral peduncle caudate-putamen cerebral cortex dorsal hypothalamic area fomix fasciculus retroflexus
Unilateral injection of FCi into the lhordc~c segments or lumbar enlargement of the spinal cord resulted in the deposit of the retrograde marker in the dorsal and ventral gray matter as well as the adjacent white matter on the side of the injection. Due to the large size of the injection, the injected FG was also found to be unevenly spread to one-third of the opposite side of the spinal cord. The distribution of FG-labeled neurons in the hypothalamus was identified in each case. The most intensely fluorescent retrogradely labeled neurons were seen ipsilateral to the injection of the tracer. At the level of the rostra1 hypothalamus, the majority of retrogradely labeled neurons were located in the parvocellular division of the paraventricular nucleus (PVN) (Fig. IC, D). A few large, FG-labeled neurons were also seen in the magnocellular division of the PVN. In addition, scattered retrogradely labeled neurons were observed in the rostrochiasmatic area. At the level caudal to the PVN, a large number of retrogradely labeled neurons were found in the lateral hypothalamic area and the zona incerta (Fig. lE--G). This group of spinally projecting neurons extends to the caudal one-third of the hypothalamus. At the more caudal level of the hypothalamus. an appreciable number of FG-labeled neurons were also found in the dorsal hypothalamic area, dorsolateral to the third ventricle and medial to the mammillothalamic tract and fornix. The distribution of the hypothalamospinal neurons has previously been extensively desc~bed,l.6,’ 1.14,23.25,26 they will not be described here in detail. The distribution of the hypothalamic NKB P2-LI neurons is shown in Fig. 1. Neurons stained for NKB-P2 were found in the nucleus arcuate, median eminence, the bed nucleus of the stria terminalis, the central nucleus and the anterior cortical nucleus of the amygdaloid complex, lateral preoptic area, and dorsal hypothalamic area. In the arcuate nucleus, a large number of neurons and fibers was found to have an intensive fluorescent signal for NKB-P2 immunostaining (Fig. 2A). Neurons and fibers having such an intensive staining for
used in the figure.7 GP IC LHA LPO LV ME ML MT oc OT ;: so VH 21
globus pallidus internal capsule lateral hypothalamic area lateral preoptic area lateral ventricle medial eminence medial lemniscus mammillothalamic tract optic chiasm optic tract reticular thalamic nucleus suprachiasmatic nucleus supraoptic nucleus ventral horn zona incerta
Hypothalamospinal
NKB-P2
Fig. lA-D
neurons
1022
H. ZHL~Oand C. J. HELKE
I”
CX
/’
Fig. IE-H
Hypothalamospinal
NKB-P2
neurons
Fig. 2. Photomicrographs of NKB P2-LI neurons in the arcuate nucleus (A), external layer of the median (B), central nucleus of the amygdaloid complex (C). and the bed nucleus of the stria terminalis (D). Scale bar in D = 60 ilrn, and applies to A--D.
eminence
NKB-P2 were also seen in the external (Fig. 2B) and internal zones of the median eminence. Neurons and fibers which were stained with NKB-P2 were also observed in the central nucleus (Fig. 2C) and anterior
cortical nucleus of the amygdaloid complex. In the bed nucleus of the stria terminalis (Fig. 2D), NKB-P2 neurons and fibers formed a well-defined oval mass. Scattered NKB P-2 neurons were also detected in the
Fig. I. (A-H) Schematic drawings of the localization of three types of neurons, FG-labeled (O), NKB P2-LI (0) and FG and NKB P2-Ll double labeled (*), in the hypothalamus of the rat. FG was retrogradely transported from thoracic segments (T,_,) of the spinal cord. For convenience, labeled neurons are indicated only on the right side of the plotting, with abbreviations on the left.
1024
H. ZHIJO
and
C.
J.
HELM
Fig. 3. Photo~crographs of NKB PZ-LI (A), and FG (B) neurons in the dorsal h~othalami~ area oi the rat. NKB PZ-LI and FG doubie-labeled neurons are indicated by arrows. Scale bar in B = 30 (2ni.
lateral preoptic area. The majority of the NKB-P2 neurons in these nuclei had a cell size of 10 x 15 pm, except the anterior cortical nucleus in which the stained neurons are smaller. In the posterior hypothalamus, many NKB PZLI neurons were found in the dorsal hypothalamic area. These NKB P2-LI neurons were dist~buted dorsoia~raIly between the third ventricle and the medial aspect of the mammillothalamic tract and fornix, and extend caudally to the periaqueductai gray of the midbrain. NKB P2-LI neurons in the dorsal h~othalamic area had a cell size of 15 x 25pm. Cells that were doubly labeled with NKB-P2 and FG were only seen in the dorsal hypothalamic area of the posterior hypothalamus following injection of the tracer into the spinal cord (Pigs 1, 3A, B). We found no differences in the distribution patterns of the FG and NKB P2-LI double-ladled neurons resulting from FG injections to the thoracic vs lumbar cord.
NKB P2-LI and FG doubly labeled neurons in the dorsal hypothalamus were of a variety of shapes (round, oral, fusiform and polygonal). FG and NKB-P2 double-labeled neurons in these areas were 15 x 25~m in size. To further study the percentage distribution of the spinally projecting NKB-PZ-LI neurons in the dorsal hypothalamic area, we counted about 1500 neurons from four rats. Whereas FGlabeled neurons were found in the other locations of the hypothalamus, because of the lack of overlapping of the FG-labeled cells with the NKB P2-LI neurons in these regions, cell counts were limited to the dorsal hypothalamic area. Cell counts showed that about 36% of the NKB-P2 neurons in the dorsal hypothalamic area projected to the t~ora~olumbar spinal cord. About 28% of the spinal projecting neurons in the dorsal hypothalamic area were positive for NKB PZ-LI {see Table 1). In the present study, the distribution of fibers and terminals which have NKB P2-LI in the spinal cord
Table 1. Ratio of spinal-projecting neurokinin B peptide-Zfike immunoreactive cells in the dorsal hypoth~amic area following FluoroGold injection into the lumbar or thoracic segments of the spinal cord FG injection site R 16 (Lumbar) R 17 (Lumbar) R 24 (Thoracic) R 25 (Thoracic)
Total (4 Rats)
FG t NKB-P2/FG = % 951449 = 521200= I 191331= 29162 =
21.2% 26.0% 35.9% 46.0%
29511042= 2813%
FG + NKB-P2/NKB-P2 = % 95/306 = 521208= 119/245 = 29150 =
3 1.O% 25% 48.6% 58.0%
29.51809= 36.5%
Cells were counted sequentially from 28 of the total 32 sections in rat 16, from 21 of the 32 sections in rat 17, from 20 of the 32 sections in rat 24, and from eight of the 32 sections in rat 25.
Hypothalamospinal of the rat was also investigated. In general, NKB P2-LI was found mainly in the superficial dorsal horn and lamina X of the spinal cord. The distribution of NKB PZLI fibers and terminals in representative sections of the third thoracic segment and the sixth lumbar segment of the spinal cord are shown diagrammatically in Fig. 4. In the superficial dorsal horn, a dense accumulation of very fine NKB PZLI fibers and granules was found in lamina II (Fig. 5A, C). Few immunoreactive fibers were detected in laminaeae I and III. Immunostained fibers and varicosities were also seen in lamina X (central autonomic area) (Fig. 5B). In laminae
NKB-P2 neurons
1025
IV and V, many immunostained fibers were seen in the intermediate gray and found to course ventromedially into lamina X. Only a few NKB P2-LI fibers and varicosities were detected in laminae VI and VII of the spinal cord. In lamina VIII and IX of the spinal cord, scattered NKB-P2-LI fibers and terminals surrounded some motoneurons (Fig. 5D). In the dorsal lateral funiculus of the spinal cord, a small number of NKB-P2 fibers were also observed. The NKB-P2 fiber pattern was consistent in all levels of the spinal cord with the highest intensity at lumbar level.
Fig. 4. Camera lucida drawing of the distribution of NKB P2-LI fibers and terminals in the third thoracic (T,) and sixth lumbar (L,) segments of the spinal cord of the rat.
1025
tt.
ZHCU
and C. .I. HELM:
Fig. 5. Photomicrographs of NKB P2-Ll fibers and terminals in the superficial dorsal horn of the thu-d thoracic segment (A) and sixth lumbar segment (C), central autonomic area (B), and lamina IX of the ventral horn (D) of the rat spinal cord. I, II, III, and IV refer to Rexed’s laminae. Asterisk in B indicates staining artifact. Scale bar in D = 60 ,~rn for A and C, and 30 pm for B and D.
Hypothalamospinal DISCUSSION
The results of the present study, using retrograde transport of FG in combination with NKB-P2 immunocytochemistry, revealed the hypothalamic distribution of NKB PZLI neurons which project to the spinal cord. The distribution of the NKB P2-LI neurons in the hypothalamic area and the distribution of NKB P2-LI fibers and varicosities in the spinal cord were also studied. Hypothalamospinal
prqiection
systems
The organization and the neurochemical nature of hypothalamospinal projections have been extensively studied in a variety of species, including cat, monkey, opossum. rabbit, hamster and rat.4 6~‘4~‘s.23.‘6 The results of the present study further confirmed previous findings that distinct groups of hypothalamic neurons, such as the paraventricular nucleus. lateral hypothalamic area and zona incerta, and the dorsal hypothalamic area of the hypothalamus, give rise to these long descending projections. Spinal projecting and non -projecting peptide -Z-like immunoreuc‘ti~e neurons
neurokinin
B
The distribution of NKB P2-LI neurons in the hypothalamus reported here, using the immunofluorescence staining method, agrees with the distribution obtained by using the peroxidaseantiperoxidase method.“,” The morphology of NKBP3 neurons in distinct nuclei of the hypothalamus was further described in the present study. Among the cell groups giving rise to the long descending projections, only the neurons in the dorsal hypothalamic area of the posterior hypothalamus were found to be labeled for NKB-P2. About 28% of the spinally projecting neurons in the dorsal hypothalamic area were NKB P2-LI. These results suggest that additional neuroactive substances may be contained within some dorsal hypothalamospinal pathways. Conversely, because only 36% of the NKB P2-LI neurons in the dorsal hypothalamic area were found to project to the thoracolumbar spinal cord, additional efferent projections of these hypothalamic NKB-P2 neurons need to be investigated. These additional neurons may project to other levels of the spinal cord and/or to various CNS regions. Distinct distribution patterns of’ neurokinin B and neurakinirl B peptide- jibers in the spinal cord NKB and NKB-P2 are both diverged from the same precursor protein. preprotachykinin B. The distribution of NKB P2-LI fibers and terminals in the
NKB-P2 neurons
1017
spinal cord was investigated in the present study. We found that the distribution pattern of NKB P2-LI fibers and terminals in the spinal cord was somewhat different from that of NKB-LI. In rats, the majority of the NKB-LI fibers and terminals were restricted to the superficial dorsal horn.“,‘* NKB P2-LI fibers and terminals were seen in the superficial dorsal horn, central autonomic area (lamina X) as well as in the intermediate gray of the dorsal spinal cord. The distinct distribution patterns of NKB-LI and NKB P2-LI fibers and terminals in the spinal cord may suggest a divergent transmission of these two peptides at the terminal field after being cleaved. or may merely reflect the greater sensitivity of the NKB-P2 antibody to label less densely innervated structures. Hypothalamic neurokinin B peptideneurons ure the main source qf‘spinal neurokinin B peptide-? It is well known that medullary raphe cell groups (magnus. pallidus, and obscurus) as well as the reticular formation contain a large number of spinalprojecting neurons. Some of these bulbospinal neurons contain SP. or NKA.‘.7.‘.‘4 However. no NKB PZ-like immunoreactive neurons were detected in the medulla in the present study (negative data were not shown). Likewise. in situ hybridization of the mRNA for NKB precursor failed to label the medullary raphe neurons.” suggesting the absence of NKB precursor in the neurons of the medulla. In the present study, no NKB P2-LI spinal interneurons were found with the method used. Also, no NKBimmunopositive cell bodies or fibers were reported in the dorsal root ganglion or the spinal root.” thus. primary afferent neurons do not appear to provide NKB innervation to the spinal cord. The results 01 the present study showed that 36% of the NKB-P3 neurons in the dorsal hypothalamic area project to the thoracolumbar spinal cord. Thus, hypothalamic NKB-P2 neurons appear to be the main source of the spinal NKB P2-LI terminals. However. the physiological role of the NKB-P2 in spinal cord needs to be further investigated. AckrlvMlrd~rmmts-This work was supported by NIH grant R01 NS34876. We thank Dr James E. Krause 01 Washington University for his generous donation of NKB peptideantiserum. I)i.rclui~wrPThe opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the DOD or the USUHS. The experiments reported herein were conducted according to the principles set forth in the “Guide for the Care and the Use of Laboratory Animals,” Insitute of Animal Resources, National Research Council, DHEW Pub. No. (NIH) 74-23.
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a cytoarchitectural,
horseradish
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H.
ZHUU
and C. J.
HELKE
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14 September 1992)
Neuroscience Vol. 52. No. 4. pp. 1019-1028. 1993 Printed in Great Britain
NEUROKININ B PEPTIDE- NEURONS PROJECT FROM THE HYPOTHALAMUS TO THE THORACOLUMBAR SPINAL CORD OF THE RAT H. ZHUO and C. J. Department
of Pharmacology,
Uniformed
HELKE*
Services University MD 20814-4799. U.S.A.
of the Health
Science,
Bethesda,
Abstract-The presence and location of CNS neurokinin B peptide-2-like immunoreactive neurons that project to the spinal cord were studied by a combination of retrograde transport of fluorescent dye (FluoroGold) and fluorescence immunocytochemistry. After injections of FluoroGold into the thoracic or lumbar segments of the rat spinal cord, serial sections of brain were stained with antisera directed against neurokinin B peptide-2. The results of the study showed that neurokinin B peptide-2-like immunoreactive neurons were located in the nucleus arcuate, median eminence, ventral and external bed nuclei of the stria terminalis, dorsal hypothalamic area, and media1 habenula. Neurokinin B peptideneurons that give rise to the long descending projections from the hypothalamus to thoracolumbar spinal cord were found only in the dorsal hypothalamic area. Approximately 36% of the neurokinin B peptideneurons in the dorsal hypothalamic area projected to the spinal cord, whereas about 28% of the spinal projecting neurons in the dorsal hypothalamic area contained neurokinin B peptide-2-like immunoreactivity. Most of the spinal projecting neurokinin B peptideneurons in the dorsal hypothalamic area had a cell size of 15 x 25 pm. In the spinal cord, immunoreactive neurokinin B peptidefibers and terminals were distributed mainly in the superficial dorsal horn and the central autonomic area, with the highest density in laminae II and X, with less density in laminae IV and V. A few neurokinin peptide-? fibers and terminals were also found in the ventral horn of the spinal cord. The results of the present study show that hypothalamic neurokinin B peptideneurons are the main source of the spinal neurokinin B peptide-2.
Mammalian namely,
tachykinins substance
substance medin
K)
and
K). These
carboxyl-terminal
include
P (SP), neurokinin
tachykinin amino
a family
neurokinin
of peptides, A (NKA,
B (NKB, peptides
acid
share
sequence
or
or
neuro-
a common Phe-X-Gly-
where X is a hydrophobic or aromatic residue.8.‘b,‘8,‘9,2’Tachykinins are widely distributed in both the CNS and peripheral tissues and evoke a variety of biological activities (for review see Refs 18, 22). Four precursors of mammalian tachykinins are encoded by two distinct genes. They are c(-, b-, and y-preprotachykinin-A which contain sequences of substance P and neurokinin A,8.‘2.20 and preprotachykinin-B which contains the sequence of neurokinin B.13 Besides NKB, the preprotachykinin B contains a 30-amino acid non-tachykinin sequence (Leu-Try-Asp-Ser-Arg-Ser-Ile-Ser-Leu-Glu-GlyLeu-Leu-Lys-Val-Leu-Ser-Lys-Ala-Ser-Val-Gly-ProLys-Glu-Thr-Ser-Leu-Pro-Gin)” called peptide 2 or NKB peptide(NKB-P2). Recently, a highly specific antiserum directed against NKB-P2 was developed by Krause ef al. ” Because of the non-tachykinin structure of NKB-P2, antibodies to NKB-P2 can be Leu-Met-NH,,
*To whom correspondence should be addressed. Abbreuiafions: FG, FluoroGold; -LI, -like immunoreactive; NGS, normal goat serum; NKA, neurokinin A; NKB, neurokinin B; NKB-P2, neurokinin B peptide-2; PBS, phosphate-buffered saline; PVN. paraventricular nucleus; SP, substance P.
valuable to identify NKB neurons and fibers without the problems of cross-reaction with tachykinin peptides. Studies using in situ hybridization and immunocytochemistry on rat” and on hamster” showed that the majority of the CNS NKB-P2 neurons are located in the hypothalamus. Preprotachykinin B, the precursor for NKB and NKB-P2, was reported to have higher levels in the hypothalamus than other regions of the CNS.‘3.‘7 Preliminary experiments of the present study showed NKB-P24ike immunoreactive (-LI) fibers and terminals mainly in the superficial dorsal horn and the central autonomic area (lamina X) of the spinal cord. This distribution was distinct from that of other tachykinin (e.g. SP, NKA) fibers and terminals.8~‘0~22~28 The present study combined retrograde transport of FluoroGold (FG) and NKB-P2 immunocytochemistry to determine the site of the origin of the CNS NKB-P2 projections to the spinal cord.
EXPERIMENTAL
PROCEDURES
Animal preparation Ten male Sprague-Dawley rats (weighing 250-450 g. Taconic Farms) were used in this study. Laminectomies of thoracic or lumbar spinal cord were performed under halothane anesthesia. One to two microliters of 4% FG (Fluorochrome Inc., U.S.A.) in saline was injected 1019
unilaterally
into the thoracic
(T,,)
enlargement FG was delivered to the spinal cord via multiple injections. attempting to involve dorsal and ventral horns of the spinal cord. so as to maximally label spinally projecting neurons. After 48-72 h survival, colchicine (60 pg in 10 ~1 of saline) was administered i.c. under halothane anesthesia (2% in oxygen). Twenty-four hours later, animals were given an overdose of chloral hydrate and perfused intracardially with the following solutions: 200ml of 100 units of heparin in saline; 400 ml of ice-cold 4% paraformaldehyde in 0. I M phosphate buffer (pH 7.4). The brain and spinal cord were removed, post-fixed in the same fixative overnight, and soaked in 30% of sucrose in 0.1 M phosphate-buffered saline (PBS) for another 24 h at 4 C.
RESUI,TS
or lumbar
(L,) of the spinal cord using a glass micropipette.
Immunohistochemistq The brain was frozen, sectioned at 25 pm with a cryostat, and tissue sections were collected in PBS. Free-floating brain sections were processed with indirect immunofluorescence staining method. After pre-incubation for 30min in 3% normal goat serum (NGS) in PBS with 0.3% Triton X-100, the sections were first incubated for 48 h with the primary antiserum, rabbit NKB-P2 (generously donated by Dr Krause, Washington University), prepared in 1% NGS in 0.1 M PBS (l:lO@O), and then incubated with the fluorescein-labeled goat anti-rabbit IgG (Jackson Lab., U.S.A.) in 0.1 M PBS (1:lOO) for 1 h at room temperature. The processed sections were wet-mounted onto glass slides and coverslipped with Permafluor Aqueous Mounting Medium (Lipshaw. U.S.A.). Sections were examined and photographed with a Leitz fluorescence photomicroscope. The spinal cord segments (injection sites) were sectioned at 60 pm thickness. The injection sites for FG in the spinal cord were identified and drawn using camera lucida. Characterisrics
of the antisera
A detailed characterization of the polyclonal antisera for NKB-P2 has been described elsewhere.” Briefly, the NKB-P2 antisera were raised against a synthetic peptide corresponding to the amino acids 5@-79 of the rat preprotachykinin B.’ It does not cross-react with substance P. NKA. NKB and neuropeptide K.” Control experiments
To ascertain whether the immunocytochemical reactions were specific. three control experiments were performed on representative sections. (I) Prior to immunostaining, the diluted antiserum was absorbed with the synthetic NKB-P2 (100 pg/ml of the final diluted antiserum). (2) The primary antiserum was omitted in the first incubation. (3) Normal rabbit serum was used instead of the primary antiserum in the first incubation. The control experiments resulted in no staining in the representative tissue sections.
Abbreviations 3v AC ACe ACo Ar BST
cc
CA3 CP CPU cx DA F FR
3rd ventricle anterior commissure central nucleus of the amygdaloid complex anterior cortical nucleus of the amygdaloid complex nucleus arcuate bed nucleus of the stria terminalis central canal hippocampus cerebral peduncle caudate-putamen cerebral cortex dorsal hypothalamic area fomix fasciculus retroflexus
Unilateral injection of FCi into the lhordc~c segments or lumbar enlargement of the spinal cord resulted in the deposit of the retrograde marker in the dorsal and ventral gray matter as well as the adjacent white matter on the side of the injection. Due to the large size of the injection, the injected FG was also found to be unevenly spread to one-third of the opposite side of the spinal cord. The distribution of FG-labeled neurons in the hypothalamus was identified in each case. The most intensely fluorescent retrogradely labeled neurons were seen ipsilateral to the injection of the tracer. At the level of the rostra1 hypothalamus, the majority of retrogradely labeled neurons were located in the parvocellular division of the paraventricular nucleus (PVN) (Fig. IC, D). A few large, FG-labeled neurons were also seen in the magnocellular division of the PVN. In addition, scattered retrogradely labeled neurons were observed in the rostrochiasmatic area. At the level caudal to the PVN, a large number of retrogradely labeled neurons were found in the lateral hypothalamic area and the zona incerta (Fig. lE--G). This group of spinally projecting neurons extends to the caudal one-third of the hypothalamus. At the more caudal level of the hypothalamus. an appreciable number of FG-labeled neurons were also found in the dorsal hypothalamic area, dorsolateral to the third ventricle and medial to the mammillothalamic tract and fornix. The distribution of the hypothalamospinal neurons has previously been extensively desc~bed,l.6,’ 1.14,23.25,26 they will not be described here in detail. The distribution of the hypothalamic NKB P2-LI neurons is shown in Fig. 1. Neurons stained for NKB-P2 were found in the nucleus arcuate, median eminence, the bed nucleus of the stria terminalis, the central nucleus and the anterior cortical nucleus of the amygdaloid complex, lateral preoptic area, and dorsal hypothalamic area. In the arcuate nucleus, a large number of neurons and fibers was found to have an intensive fluorescent signal for NKB-P2 immunostaining (Fig. 2A). Neurons and fibers having such an intensive staining for
used in the figure.7 GP IC LHA LPO LV ME ML MT oc OT ;: so VH 21
globus pallidus internal capsule lateral hypothalamic area lateral preoptic area lateral ventricle medial eminence medial lemniscus mammillothalamic tract optic chiasm optic tract reticular thalamic nucleus suprachiasmatic nucleus supraoptic nucleus ventral horn zona incerta
Hypothalamospinal
NKB-P2
Fig. lA-D
neurons
1022
H. ZHL~Oand C. J. HELKE
I”
CX
/’
Fig. IE-H
Hypothalamospinal
NKB-P2
neurons
Fig. 2. Photomicrographs of NKB P2-LI neurons in the arcuate nucleus (A), external layer of the median (B), central nucleus of the amygdaloid complex (C). and the bed nucleus of the stria terminalis (D). Scale bar in D = 60 ilrn, and applies to A--D.
eminence
NKB-P2 were also seen in the external (Fig. 2B) and internal zones of the median eminence. Neurons and fibers which were stained with NKB-P2 were also observed in the central nucleus (Fig. 2C) and anterior
cortical nucleus of the amygdaloid complex. In the bed nucleus of the stria terminalis (Fig. 2D), NKB-P2 neurons and fibers formed a well-defined oval mass. Scattered NKB P-2 neurons were also detected in the
Fig. I. (A-H) Schematic drawings of the localization of three types of neurons, FG-labeled (O), NKB P2-LI (0) and FG and NKB P2-Ll double labeled (*), in the hypothalamus of the rat. FG was retrogradely transported from thoracic segments (T,_,) of the spinal cord. For convenience, labeled neurons are indicated only on the right side of the plotting, with abbreviations on the left.
1024
H. ZHIJO
and
C.
J.
HELM
Fig. 3. Photo~crographs of NKB PZ-LI (A), and FG (B) neurons in the dorsal h~othalami~ area oi the rat. NKB PZ-LI and FG doubie-labeled neurons are indicated by arrows. Scale bar in B = 30 (2ni.
lateral preoptic area. The majority of the NKB-P2 neurons in these nuclei had a cell size of 10 x 15 pm, except the anterior cortical nucleus in which the stained neurons are smaller. In the posterior hypothalamus, many NKB PZLI neurons were found in the dorsal hypothalamic area. These NKB P2-LI neurons were dist~buted dorsoia~raIly between the third ventricle and the medial aspect of the mammillothalamic tract and fornix, and extend caudally to the periaqueductai gray of the midbrain. NKB P2-LI neurons in the dorsal h~othalamic area had a cell size of 15 x 25pm. Cells that were doubly labeled with NKB-P2 and FG were only seen in the dorsal hypothalamic area of the posterior hypothalamus following injection of the tracer into the spinal cord (Pigs 1, 3A, B). We found no differences in the distribution patterns of the FG and NKB P2-LI double-ladled neurons resulting from FG injections to the thoracic vs lumbar cord.
NKB P2-LI and FG doubly labeled neurons in the dorsal hypothalamus were of a variety of shapes (round, oral, fusiform and polygonal). FG and NKB-P2 double-labeled neurons in these areas were 15 x 25~m in size. To further study the percentage distribution of the spinally projecting NKB-PZ-LI neurons in the dorsal hypothalamic area, we counted about 1500 neurons from four rats. Whereas FGlabeled neurons were found in the other locations of the hypothalamus, because of the lack of overlapping of the FG-labeled cells with the NKB P2-LI neurons in these regions, cell counts were limited to the dorsal hypothalamic area. Cell counts showed that about 36% of the NKB-P2 neurons in the dorsal hypothalamic area projected to the t~ora~olumbar spinal cord. About 28% of the spinal projecting neurons in the dorsal hypothalamic area were positive for NKB PZ-LI {see Table 1). In the present study, the distribution of fibers and terminals which have NKB P2-LI in the spinal cord
Table 1. Ratio of spinal-projecting neurokinin B peptide-Zfike immunoreactive cells in the dorsal hypoth~amic area following FluoroGold injection into the lumbar or thoracic segments of the spinal cord FG injection site R 16 (Lumbar) R 17 (Lumbar) R 24 (Thoracic) R 25 (Thoracic)
Total (4 Rats)
FG t NKB-P2/FG = % 951449 = 521200= I 191331= 29162 =
21.2% 26.0% 35.9% 46.0%
29511042= 2813%
FG + NKB-P2/NKB-P2 = % 95/306 = 521208= 119/245 = 29150 =
3 1.O% 25% 48.6% 58.0%
29.51809= 36.5%
Cells were counted sequentially from 28 of the total 32 sections in rat 16, from 21 of the 32 sections in rat 17, from 20 of the 32 sections in rat 24, and from eight of the 32 sections in rat 25.
Hypothalamospinal of the rat was also investigated. In general, NKB P2-LI was found mainly in the superficial dorsal horn and lamina X of the spinal cord. The distribution of NKB PZLI fibers and terminals in representative sections of the third thoracic segment and the sixth lumbar segment of the spinal cord are shown diagrammatically in Fig. 4. In the superficial dorsal horn, a dense accumulation of very fine NKB PZLI fibers and granules was found in lamina II (Fig. 5A, C). Few immunoreactive fibers were detected in laminaeae I and III. Immunostained fibers and varicosities were also seen in lamina X (central autonomic area) (Fig. 5B). In laminae
NKB-P2 neurons
1025
IV and V, many immunostained fibers were seen in the intermediate gray and found to course ventromedially into lamina X. Only a few NKB P2-LI fibers and varicosities were detected in laminae VI and VII of the spinal cord. In lamina VIII and IX of the spinal cord, scattered NKB-P2-LI fibers and terminals surrounded some motoneurons (Fig. 5D). In the dorsal lateral funiculus of the spinal cord, a small number of NKB-P2 fibers were also observed. The NKB-P2 fiber pattern was consistent in all levels of the spinal cord with the highest intensity at lumbar level.
Fig. 4. Camera lucida drawing of the distribution of NKB P2-LI fibers and terminals in the third thoracic (T,) and sixth lumbar (L,) segments of the spinal cord of the rat.
1025
tt.
ZHCU
and C. .I. HELM:
Fig. 5. Photomicrographs of NKB P2-Ll fibers and terminals in the superficial dorsal horn of the thu-d thoracic segment (A) and sixth lumbar segment (C), central autonomic area (B), and lamina IX of the ventral horn (D) of the rat spinal cord. I, II, III, and IV refer to Rexed’s laminae. Asterisk in B indicates staining artifact. Scale bar in D = 60 ,~rn for A and C, and 30 pm for B and D.
Hypothalamospinal DISCUSSION
The results of the present study, using retrograde transport of FG in combination with NKB-P2 immunocytochemistry, revealed the hypothalamic distribution of NKB PZLI neurons which project to the spinal cord. The distribution of the NKB P2-LI neurons in the hypothalamic area and the distribution of NKB P2-LI fibers and varicosities in the spinal cord were also studied. Hypothalamospinal
prqiection
systems
The organization and the neurochemical nature of hypothalamospinal projections have been extensively studied in a variety of species, including cat, monkey, opossum. rabbit, hamster and rat.4 6~‘4~‘s.23.‘6 The results of the present study further confirmed previous findings that distinct groups of hypothalamic neurons, such as the paraventricular nucleus. lateral hypothalamic area and zona incerta, and the dorsal hypothalamic area of the hypothalamus, give rise to these long descending projections. Spinal projecting and non -projecting peptide -Z-like immunoreuc‘ti~e neurons
neurokinin
B
The distribution of NKB P2-LI neurons in the hypothalamus reported here, using the immunofluorescence staining method, agrees with the distribution obtained by using the peroxidaseantiperoxidase method.“,” The morphology of NKBP3 neurons in distinct nuclei of the hypothalamus was further described in the present study. Among the cell groups giving rise to the long descending projections, only the neurons in the dorsal hypothalamic area of the posterior hypothalamus were found to be labeled for NKB-P2. About 28% of the spinally projecting neurons in the dorsal hypothalamic area were NKB P2-LI. These results suggest that additional neuroactive substances may be contained within some dorsal hypothalamospinal pathways. Conversely, because only 36% of the NKB P2-LI neurons in the dorsal hypothalamic area were found to project to the thoracolumbar spinal cord, additional efferent projections of these hypothalamic NKB-P2 neurons need to be investigated. These additional neurons may project to other levels of the spinal cord and/or to various CNS regions. Distinct distribution patterns of’ neurokinin B and neurakinirl B peptide- jibers in the spinal cord NKB and NKB-P2 are both diverged from the same precursor protein. preprotachykinin B. The distribution of NKB P2-LI fibers and terminals in the
NKB-P2 neurons
1017
spinal cord was investigated in the present study. We found that the distribution pattern of NKB P2-LI fibers and terminals in the spinal cord was somewhat different from that of NKB-LI. In rats, the majority of the NKB-LI fibers and terminals were restricted to the superficial dorsal horn.“,‘* NKB P2-LI fibers and terminals were seen in the superficial dorsal horn, central autonomic area (lamina X) as well as in the intermediate gray of the dorsal spinal cord. The distinct distribution patterns of NKB-LI and NKB P2-LI fibers and terminals in the spinal cord may suggest a divergent transmission of these two peptides at the terminal field after being cleaved. or may merely reflect the greater sensitivity of the NKB-P2 antibody to label less densely innervated structures. Hypothalamic neurokinin B peptideneurons ure the main source qf‘spinal neurokinin B peptide-? It is well known that medullary raphe cell groups (magnus. pallidus, and obscurus) as well as the reticular formation contain a large number of spinalprojecting neurons. Some of these bulbospinal neurons contain SP. or NKA.‘.7.‘.‘4 However. no NKB PZ-like immunoreactive neurons were detected in the medulla in the present study (negative data were not shown). Likewise. in situ hybridization of the mRNA for NKB precursor failed to label the medullary raphe neurons.” suggesting the absence of NKB precursor in the neurons of the medulla. In the present study, no NKB P2-LI spinal interneurons were found with the method used. Also, no NKBimmunopositive cell bodies or fibers were reported in the dorsal root ganglion or the spinal root.” thus. primary afferent neurons do not appear to provide NKB innervation to the spinal cord. The results 01 the present study showed that 36% of the NKB-P3 neurons in the dorsal hypothalamic area project to the thoracolumbar spinal cord. Thus, hypothalamic NKB-P2 neurons appear to be the main source of the spinal NKB P2-LI terminals. However. the physiological role of the NKB-P2 in spinal cord needs to be further investigated. AckrlvMlrd~rmmts-This work was supported by NIH grant R01 NS34876. We thank Dr James E. Krause 01 Washington University for his generous donation of NKB peptideantiserum. I)i.rclui~wrPThe opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the DOD or the USUHS. The experiments reported herein were conducted according to the principles set forth in the “Guide for the Care and the Use of Laboratory Animals,” Insitute of Animal Resources, National Research Council, DHEW Pub. No. (NIH) 74-23.
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