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Prenatal ontogeny of substance P-like immunoreactivity in the parabrachial nucleus of the human fetus — an immunocytochemical study
316
Bra#~ Research, 590 ( i 992) 316-320 ~ It}92 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 25347
Prenatal ontogeny of substance P-like immunoreactivity in the parabrachial nucleus of the human fetus an immunocytochemical study Li G e n W a n g , Z h i R e n R a o a n d Ji Shuo Li Department of Anatomy. Fourth Milita~' Medical Unirersity, Xian (P.R. China) (Accepted 16 June 1992)
Key words: Substance P: Immunocytochemistry; Ontogeny; Parabrachial nucleus; Human fetus
Using an immunocytochemical method, the prenatal ontogeny of substance P-like immunoreactivity (SP-LI) was demonstrated in the parabrachial nucleus (PB) of human Ictus. fetal age (menstruation age) 11.5 to 40 weeks. The time of initial appearance of SP-L! in the human brainstem PB was between fetal age 1!.5 weeks and 16 weeks. While the fetus grew, the density of SP-L! fibers and terminals in the human fetus brainstem PB increased constantly from fetal age 16 weeks to 40 weeks. These findings indicate that substance P (SP) might play an important role in the human parabrachial nucleus development and in its functional establishment during the prenatal period.
The synaptically active agent substance P (SP) -'a belongs to a newly identified class of peptidergic neurotransmitters or modulators '~''M which participate in different functional systems in the CNS. However, the comprehensive understanding of its function without studying of the ontogenetic aspects would be impossible. Studies on the development of SP in the CNS of rat '~, mice ~4 and man 4't~ revealed interesting information. These studies showed that SP made its initial appearance during the early prenatal period, and proposed that SP may play an important role for the early ontogenetic development of the CNS. Unfortunately, studies of the ontogenetic distribution of SP in the human CNS were fragmentary, simply because human fetuses were difficult to obtain 4't5. In order to give a whole idea of the prenatal ontogeny of SP-like immunoreactivity (SP-LI) in the human brainstem, consecutive with our previous reports 2~, by using the same materials, this study was carried out to reveal the ontogenetic distribution and development of SP-LI in the parabrachial nucleus (PB) of human fetuses and to add some information on the understanding of SP. Seven human fetuses of both sexes that died of spontaneous abortion without any disease involving the
CNS were obtained. Estimation of the fetal age was done according to the last menstruation data and with reference to the measurement of crown-rump length and body weight ~', For detailed information of the materials, see Table i. Within I-4 h after abortion and death, the fetuses were transcardiaUy perfuscd with an adeqate amount of normal saline (0.9% sodium chloride) followed by Zamboni's fixitive 31~, and then the brain was immersed in phosphate buffer (0.01 M, pH 7.4) containing 30% sucrose, for 24-72 h at 4"C. Serial frontal frozen sections were cut on a Leitz Cryotome with the section thickness ranging between 40 and 60 btm,
One fourth of the sections were subjected to immunocytochemistry according to Hsu's ABC method 7, with the primary antibody against SP. Before immunocytochemistry, sections were put in methanol containing 0.1% H20, for 30 rain at room temperature to prevent demonstration of endogenous peroxidase activity in the blood cells t2 and then put in a buffer (0.01 M, pH 7.4) containing 0.3% bovine albumin and 0.01% Triton X-100 at 37°C for 30 rain. Sections were incubated with rabbit anti-SP antiserum (1"10,000) in a humid atmosphere at 37°C for 30 rain and then moved
Correslmmlence: L.G. WanG, Department of Anatomy, Fourth Military Medical University, Xian 710032, Shaanxi Province, P.R. China.
317 TABLE ! Material conditions No.
Fetal age
Sex
Weight
Crown - nmlp length
3-44 3-41 3-30 3-36 3-11 3-17 1- l I
11.5 weeks 16 weeks 23 weeks 27 weeks 31 weeks 35 weeks 40 weeks
male male female male female male male
25 g 40g 700 g 950 g 1500 g 3000g 3 400 g
9.5 cm 13.0cm 21.0 cm 18.5 cm 18.0 cm 22.0cm 35.0 cm
to a refrigerator at 4°C for 24 h, and then incubated with biotinized donkey anti-rabbit IgG (Amersham, UK, 1:300). Then sections were incubated with ABC complex (Amersham, UK; 1:200) at 37°C for 30 rain and then moved to a refrigerator at 4°C for 24 h. After that, sections were processed according to Shu's method 27 to reveal peroxidase activity incorporated in the ABC complex, and then mounted onto gelatinized slides and coverslipped with neutral balsam. Some of the sections were incubated with control serum which was obtained by presbsorption of the rabbit anti-SP with excessive SP (Belgium Bioproduction, 100 ~ g / m l ) . One fourth of the sections was stained with Cresyl violet for normal structure identification. The control sections revealed negative staining. The PB has traditionally been divided into medial (pbm) and lateral (pbl) divisions and a ventrolateral extension, the KoUiker-Fuse nucleus (KF), on the ba-
sis of the description by Olszewski and Baxter of the human brainstem 2°. We used the same terminology to describe our results. The smallest fetus in this study was one of 11.5 weeks of age, its brainstem was developing, the internal structures and external shape were still primitive as compared with those of other older fetuses. No SP-LI structures were demonstrated in the PB of this brainstem. When the fetal age was 16 weeks, the brainstem had developed to a relatively more mature state. There were low densities of SP-LI fibers and terminals in pbm, phi and KF nucleus (Fig. IA). The fibers were thin and smooth, with a few varicosities (Fig. 1B). On these terminals there were few terminal boutons (Fig.
1a). Reaching the fetal age of 23 weeks, the brainstem went on growing and the density of SP-LI structures increased remarkably. In pbl there were moderate to
Fig. 1. A: photomicrograph of the left PB of the 16 week fetus, showing the distribution of SP-LI fibers and terminals distributed in it. The BC itself showed no SP-LI structures. Bar -- 150 ~m. B: a magnification photograph of the blocked area in A, showing the SP-LI fibers and terminals distributed in the pbl. The arrows indicate a fine SP-LI fibers with few varicosities on it. Bar -- 15 ~m. pbi, lateral parabrachial nucleus; pbm, medial parabrachial nucleus; BC, brachium conjunctiva: KF, Kolliker-Fuse nucleus.
318 No SP-LI cell was found in the PB of the fetuses studied. Previous studies 4'Ls of the ontogeny of SP-LI structures in the human brain have not revealed its initial time of appearance• Although the present study could not demonstrate the exact time of initial appearance, there is one thing that we are sure of: the initial time of appearance of SP-LI in the human brainstem was between the fetal age of 11.5 weeks and 16 weeks. The PB was among the early expression sites of SP-LI structures in the brainstem. Earlier studies on the parabrachial nucleus were intrigued vy the findings of Norgren ~aTa~ that the medial parabrachial nucleus receives most of the efferent projections from the oral segment of the nucleus tractus solitari, namely the gustatory information, the parabrachial nucleus assumed to be an important relay station for gustatory information to the forebrain. Later studies indicated that the nucleus tractus solitarii, which relays general visceral afferents from cardiac, pulmonary and gastrointestinal afferents, projects mainly to the lateral parabrachial nucleus and Koiliker-Fuse nucleus, and then from here projects again to forebrain ~'~°'~n'ua'ns'22.PB also received input from the central nucleus of the amygdala", area postrema ~ and
high densities of SP-LI fibers and terminals, and also in pbm and the KF nucleus there were moderate densities of SP-L! fibers and terminals. The brachium conjunctiva (BC) showed no SP-L! fibers, but in some instances some SP-LI fiber bundles stretched in to BC from pbl and pbm, interrupting the opaque area where the BC locates (Fig. 2A). On these fibers there were lots of varicosities, and the diameters of these fibers became larger that that at the fetal age 16 weeks (Fig. 2B). On the end of these terminals many terminal boutons were detected. From fetal age 27 weeks on, until fetal age 40 weeks, the histological development of the brainstem went on even more rapidly and the mass augmentation of the brainstem was obvious. The distribution patterns of SP-L! structures in the PB complex showed little changes, but the density of SP-LI structures in the PB increased consistently. When the fetal age reached 40 weeks, the density of SP-LI fibers and terminals was the highe.~t among the fetuses studied (Fig. 3A), and the diameters of these fibers were larger than that of other fetus (measured simply by applying the micromeasurement device equiped on the eye-piece of the Olympus BH-2 light microscope), and with more varicosities on them (Fig. 3B).
,
'
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7'
+41
p!
al
O h,
.
•
. ,
, ;:,.. -2 .°
, Op
t
,,+~"
Ib',~ 1 ,
I1' +
j •
•
"¢, i
f i
• ~°
Fig. 2, A: photomicrograph of the left PB of the 23 weeks fetus, showing the distribution of SP-L! fibers and terminals distributed in it. The BC was interupted by bundles of SP-LI fibers stretching from both the pbl and pbm. Bar -~. 150 ~,m, B: a magnification of the blocked area in A, showing the SP-L! fibers and terminals distributed in pbl. The density of SP-LI fibers and terminals was extremely high. Bar = 30/zm. pbl, lateral parabrachial nucleus: pbm, medial parabrachial nucleus; BC, brachium conjunctiva; KF, Kolliker-Fuse nucleus.
319
A
;o
.J
~
i 7"
BC .-,:~ ..,,.:~
jr
!
n
,ll .o
I"
'
~ p
',
o
e!
,
,"
:,,:
o
f KF Fig. 3. A: photomicrograph of the left PB of the 40 week fetus, showing the distribution of SP-L! fibers and terminals distributed in it. The BC was interupted by bundles of SP-LI fibers stretching from both the pbl and pbm. Bar ,= 150 p.m. B: a magnification photograph of the blocked area in A, showing the SP-LI fibers and terminals distributed in pbl. The density of SP-L! fibers and terminals was extremely high. Bar = 15 ~m. pbl, lateral parabrachial nucleus; pbm, medial parabrachial nucleus; BC, brachium conjunctiva; KF, Kolliker-Fuse nucleus.
lateral hypothalamic area 2. Neurons regulating respiratory -~;3, cardiovascular s and gastrointestinal tract 24 activities were located in the lateral parabrachial nucleus. It would be appropriate to postulate that SP might be an important factor for the development of the human brainstem PB nucleus and its related systems.
1 Beckstead, R.M., Morse, J.R. and Norgren, R., The nucleus of solitary tract in the monkey: projections to the thalamus and brainstem nuclei, J. Comp. Neuroi., 190 (1980) 259-282. 2 Berk, M.L. and Finkelstein, J.A., Efferent connections of the lateral hypothallamic area of the rat: an autoradiographic investigation, Brain Res. Bull., 8 (1982)511-526. 3 Betrand, F. and Hugelin, A., Respiratory synchronizing functions of the nucleus parabrachialis medialis: pneumotaxic mechanism, J. NeurophysioL, 34 (1971) 189-207, 4 Del Fiacco, M,, Dessi, M.L., Atzori, M.G. and Levanti, M.C., Substance P in the human brain stem. Preliminary results of its immunohistochemical localization 264 (1983) 142-147. 5 Hamilton, R.B., Ellenberger, H., Liskowski, D. and Schneiderman, N., Parabrachiai area as mediator of brachycardia in the rabbits, J. Auton. Nerv. Syst., 4 (1981) 261-281. 6 Hamilton, W.J. and Mossman, H.W., Human embryology: prena-
tal development of form and function, in: Growth of the Eml~tyo and Fetus; Dez'elopment of External Forms; Exthnation of Embty. onic and Fetal Age; 4th edn., W. Heifers, Cambridge, 1972, pp.
174-191. 7 Hsu, S.M., Raine, L. and Fanger, H., The use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase technique: a comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem, 29 (1981) 575-578. 8 Krettek, J.E. and Price, J.L., Amygdaloid projections to subcortical structures within the basal forebrain and brainstem in the rat and cat J. Comp. Neurol., 178 (1978)225-254. 9 Ljungdahl, A., H6kfelt, T. and Nilsson, G., Distribu~tion of substance P-like immunoreactivity in the central nervous system of rat--l, Cell bodies and nerve terminals, Neuroscience, 3 (1978) 681-943. 10 Loewy, A,D. and Burton, H., Nuclei of the solitary tract: efferent projections to the lower brainstem and spinal cord oT the cat, J. Comp. Neurol. 181 (1978) 421-450. I1 Loewy, A.D. and McKelar, S., Neuroanatomical basiis of central cardiovascular control, Fed. Prec., 39 (1980) 2459-2503. 12 Mizukawa, K., Me(Jeer, P.L., Tago, H., Peng, J.H., McGeer, E.G. and Kiinura, H., The cholinergic system of the human hindbrain studies by choline aceltransferase immunohistochemistry and acetylcholine histochemistry, Brain Res., 379 (1986) 39-55, 13 Mraovitch, S., Kumada, M. and Reis, D.S., Role of the nucleus parabrachialis cardiovascular regulation in cat, Brain Res., 232 (1982) 57-75.
320 14 Ni, L. and Jonakait, G.M., Development of substance P-containing neurons in the central nervous system in mice: an immunocytochemical study, J. Comp. NeuroL, 275 (1988)493-510. 15 Nomura, H., Shiosaka, S., Inagaki, S., lnagaki, S., Ishimoto, 1., Senba, E., Sakanaka, M,, Takatsuki, K., Matsukaki, T., Kubota, Y,, Saito, S., Kagure, K. and Tohyama, M., Distribution of substance P-like immunoreactivity in the lower brainstem of human fetus: an immunohistochemical study, Brain Res., 252 (1982) 315-325. 16 Norgren, R,, Gustatory afferents to ventral forebrain, Brain Res., 81 (1974) 285-295. 17 Norgren, R., Taste pathways to hypothalamus and amygdala, 3, Comp. Neurol., 166 (1976) 17-30. 18 Norgren, R., Projections from the nucleus of the solitary tract in the rat, Neuroscience, 3 (1978) 207-218. 19 Norgren, R. and Leonard, C.M., Ascending central gustatory connections, J. Comp. Neurol., 150 (1973)217-283. 20 OIszewski, J. and Baxter, D., Cytoarchitecture of tile Human Brain Swm, Lippincott, Philadelphia, 1954. 21 Pernow, B., Substance P, Pharmacol Rel'., 35 (1983) 85-141. 22 Richardo, J.A., and Koh, E.T., Anatomical evidence of direct projections from the nucleus of the solitary tract to the hypothalamus, amygdala, and other forebrain structures in the rat, Braitz R,'s., 152 (1978) 1-26. 23 Riche, D., Denavit-Saubie, N. and Champagnat, J., Pontine afferents to the medullary respiratory systems: anatomicofunctional correlation, Neurosci. Lett., 13 (1979) 151-155.
24 Rogers, R.C. and Novin, D., The neurology of hepatic osmoregulation and its implications for studies of ingestive behaviour, Brain Res. Bull., Suppl. 4, 5 (1980) 189-194. 25 Sakanaka, M., Shiosaka, S., Takatsuki, K., Tagaki, H., Senba, S., Kawai, E., Matsuzaki, T. and Tohyama, M., Ontogeny of substance P-containing neuron system of the rat: immunohistochemical analysis-ll. Lower brain stem Neuroscience, 7 (1982) 10971126. 26 Shapiro, R.E. and Miselis, R.R., An afferent projections from the area postrema and the caudal medial nucleus of the solitary tract to the parabrachial nucleus of the rat Soc. Neurosci. Abstr., 8 (I 982) 269. 27 Shu, S., Ju, G. and Fan, L., The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system, Neurosci. Lett., 85 (1988) 169-171. 28 Snyder, S.H., Brain peptides and neurotransmitters, Science, 209 (1980) 976-983. 29 Wang, L.G., Rap, Z.R. and Li, J.S., Prenatal ontogeny of substance P-like immunoreactivity in the trigeminal spinal caudal subnucleus of the human fetus - an immunocytochemical study, Brain Res., 579 (1992) 157-160. 30 ZambonL L. and de Martino, C., Buffered picric-acid formaldehyde: a new rapid fixative for electron microscopy J. CeU Biol., 35 (1967) 148A.
Bra#~ Research, 590 ( i 992) 316-320 ~ It}92 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 25347
Prenatal ontogeny of substance P-like immunoreactivity in the parabrachial nucleus of the human fetus an immunocytochemical study Li G e n W a n g , Z h i R e n R a o a n d Ji Shuo Li Department of Anatomy. Fourth Milita~' Medical Unirersity, Xian (P.R. China) (Accepted 16 June 1992)
Key words: Substance P: Immunocytochemistry; Ontogeny; Parabrachial nucleus; Human fetus
Using an immunocytochemical method, the prenatal ontogeny of substance P-like immunoreactivity (SP-LI) was demonstrated in the parabrachial nucleus (PB) of human Ictus. fetal age (menstruation age) 11.5 to 40 weeks. The time of initial appearance of SP-L! in the human brainstem PB was between fetal age 1!.5 weeks and 16 weeks. While the fetus grew, the density of SP-L! fibers and terminals in the human fetus brainstem PB increased constantly from fetal age 16 weeks to 40 weeks. These findings indicate that substance P (SP) might play an important role in the human parabrachial nucleus development and in its functional establishment during the prenatal period.
The synaptically active agent substance P (SP) -'a belongs to a newly identified class of peptidergic neurotransmitters or modulators '~''M which participate in different functional systems in the CNS. However, the comprehensive understanding of its function without studying of the ontogenetic aspects would be impossible. Studies on the development of SP in the CNS of rat '~, mice ~4 and man 4't~ revealed interesting information. These studies showed that SP made its initial appearance during the early prenatal period, and proposed that SP may play an important role for the early ontogenetic development of the CNS. Unfortunately, studies of the ontogenetic distribution of SP in the human CNS were fragmentary, simply because human fetuses were difficult to obtain 4't5. In order to give a whole idea of the prenatal ontogeny of SP-like immunoreactivity (SP-LI) in the human brainstem, consecutive with our previous reports 2~, by using the same materials, this study was carried out to reveal the ontogenetic distribution and development of SP-LI in the parabrachial nucleus (PB) of human fetuses and to add some information on the understanding of SP. Seven human fetuses of both sexes that died of spontaneous abortion without any disease involving the
CNS were obtained. Estimation of the fetal age was done according to the last menstruation data and with reference to the measurement of crown-rump length and body weight ~', For detailed information of the materials, see Table i. Within I-4 h after abortion and death, the fetuses were transcardiaUy perfuscd with an adeqate amount of normal saline (0.9% sodium chloride) followed by Zamboni's fixitive 31~, and then the brain was immersed in phosphate buffer (0.01 M, pH 7.4) containing 30% sucrose, for 24-72 h at 4"C. Serial frontal frozen sections were cut on a Leitz Cryotome with the section thickness ranging between 40 and 60 btm,
One fourth of the sections were subjected to immunocytochemistry according to Hsu's ABC method 7, with the primary antibody against SP. Before immunocytochemistry, sections were put in methanol containing 0.1% H20, for 30 rain at room temperature to prevent demonstration of endogenous peroxidase activity in the blood cells t2 and then put in a buffer (0.01 M, pH 7.4) containing 0.3% bovine albumin and 0.01% Triton X-100 at 37°C for 30 rain. Sections were incubated with rabbit anti-SP antiserum (1"10,000) in a humid atmosphere at 37°C for 30 rain and then moved
Correslmmlence: L.G. WanG, Department of Anatomy, Fourth Military Medical University, Xian 710032, Shaanxi Province, P.R. China.
317 TABLE ! Material conditions No.
Fetal age
Sex
Weight
Crown - nmlp length
3-44 3-41 3-30 3-36 3-11 3-17 1- l I
11.5 weeks 16 weeks 23 weeks 27 weeks 31 weeks 35 weeks 40 weeks
male male female male female male male
25 g 40g 700 g 950 g 1500 g 3000g 3 400 g
9.5 cm 13.0cm 21.0 cm 18.5 cm 18.0 cm 22.0cm 35.0 cm
to a refrigerator at 4°C for 24 h, and then incubated with biotinized donkey anti-rabbit IgG (Amersham, UK, 1:300). Then sections were incubated with ABC complex (Amersham, UK; 1:200) at 37°C for 30 rain and then moved to a refrigerator at 4°C for 24 h. After that, sections were processed according to Shu's method 27 to reveal peroxidase activity incorporated in the ABC complex, and then mounted onto gelatinized slides and coverslipped with neutral balsam. Some of the sections were incubated with control serum which was obtained by presbsorption of the rabbit anti-SP with excessive SP (Belgium Bioproduction, 100 ~ g / m l ) . One fourth of the sections was stained with Cresyl violet for normal structure identification. The control sections revealed negative staining. The PB has traditionally been divided into medial (pbm) and lateral (pbl) divisions and a ventrolateral extension, the KoUiker-Fuse nucleus (KF), on the ba-
sis of the description by Olszewski and Baxter of the human brainstem 2°. We used the same terminology to describe our results. The smallest fetus in this study was one of 11.5 weeks of age, its brainstem was developing, the internal structures and external shape were still primitive as compared with those of other older fetuses. No SP-LI structures were demonstrated in the PB of this brainstem. When the fetal age was 16 weeks, the brainstem had developed to a relatively more mature state. There were low densities of SP-LI fibers and terminals in pbm, phi and KF nucleus (Fig. IA). The fibers were thin and smooth, with a few varicosities (Fig. 1B). On these terminals there were few terminal boutons (Fig.
1a). Reaching the fetal age of 23 weeks, the brainstem went on growing and the density of SP-LI structures increased remarkably. In pbl there were moderate to
Fig. 1. A: photomicrograph of the left PB of the 16 week fetus, showing the distribution of SP-LI fibers and terminals distributed in it. The BC itself showed no SP-LI structures. Bar -- 150 ~m. B: a magnification photograph of the blocked area in A, showing the SP-LI fibers and terminals distributed in the pbl. The arrows indicate a fine SP-LI fibers with few varicosities on it. Bar -- 15 ~m. pbi, lateral parabrachial nucleus; pbm, medial parabrachial nucleus; BC, brachium conjunctiva: KF, Kolliker-Fuse nucleus.
318 No SP-LI cell was found in the PB of the fetuses studied. Previous studies 4'Ls of the ontogeny of SP-LI structures in the human brain have not revealed its initial time of appearance• Although the present study could not demonstrate the exact time of initial appearance, there is one thing that we are sure of: the initial time of appearance of SP-LI in the human brainstem was between the fetal age of 11.5 weeks and 16 weeks. The PB was among the early expression sites of SP-LI structures in the brainstem. Earlier studies on the parabrachial nucleus were intrigued vy the findings of Norgren ~aTa~ that the medial parabrachial nucleus receives most of the efferent projections from the oral segment of the nucleus tractus solitari, namely the gustatory information, the parabrachial nucleus assumed to be an important relay station for gustatory information to the forebrain. Later studies indicated that the nucleus tractus solitarii, which relays general visceral afferents from cardiac, pulmonary and gastrointestinal afferents, projects mainly to the lateral parabrachial nucleus and Koiliker-Fuse nucleus, and then from here projects again to forebrain ~'~°'~n'ua'ns'22.PB also received input from the central nucleus of the amygdala", area postrema ~ and
high densities of SP-LI fibers and terminals, and also in pbm and the KF nucleus there were moderate densities of SP-L! fibers and terminals. The brachium conjunctiva (BC) showed no SP-L! fibers, but in some instances some SP-LI fiber bundles stretched in to BC from pbl and pbm, interrupting the opaque area where the BC locates (Fig. 2A). On these fibers there were lots of varicosities, and the diameters of these fibers became larger that that at the fetal age 16 weeks (Fig. 2B). On the end of these terminals many terminal boutons were detected. From fetal age 27 weeks on, until fetal age 40 weeks, the histological development of the brainstem went on even more rapidly and the mass augmentation of the brainstem was obvious. The distribution patterns of SP-L! structures in the PB complex showed little changes, but the density of SP-LI structures in the PB increased consistently. When the fetal age reached 40 weeks, the density of SP-LI fibers and terminals was the highe.~t among the fetuses studied (Fig. 3A), and the diameters of these fibers were larger than that of other fetus (measured simply by applying the micromeasurement device equiped on the eye-piece of the Olympus BH-2 light microscope), and with more varicosities on them (Fig. 3B).
,
'
' +i ,;*•
7'
+41
p!
al
O h,
.
•
. ,
, ;:,.. -2 .°
, Op
t
,,+~"
Ib',~ 1 ,
I1' +
j •
•
"¢, i
f i
• ~°
Fig. 2, A: photomicrograph of the left PB of the 23 weeks fetus, showing the distribution of SP-L! fibers and terminals distributed in it. The BC was interupted by bundles of SP-LI fibers stretching from both the pbl and pbm. Bar -~. 150 ~,m, B: a magnification of the blocked area in A, showing the SP-L! fibers and terminals distributed in pbl. The density of SP-LI fibers and terminals was extremely high. Bar = 30/zm. pbl, lateral parabrachial nucleus: pbm, medial parabrachial nucleus; BC, brachium conjunctiva; KF, Kolliker-Fuse nucleus.
319
A
;o
.J
~
i 7"
BC .-,:~ ..,,.:~
jr
!
n
,ll .o
I"
'
~ p
',
o
e!
,
,"
:,,:
o
f KF Fig. 3. A: photomicrograph of the left PB of the 40 week fetus, showing the distribution of SP-L! fibers and terminals distributed in it. The BC was interupted by bundles of SP-LI fibers stretching from both the pbl and pbm. Bar ,= 150 p.m. B: a magnification photograph of the blocked area in A, showing the SP-LI fibers and terminals distributed in pbl. The density of SP-L! fibers and terminals was extremely high. Bar = 15 ~m. pbl, lateral parabrachial nucleus; pbm, medial parabrachial nucleus; BC, brachium conjunctiva; KF, Kolliker-Fuse nucleus.
lateral hypothalamic area 2. Neurons regulating respiratory -~;3, cardiovascular s and gastrointestinal tract 24 activities were located in the lateral parabrachial nucleus. It would be appropriate to postulate that SP might be an important factor for the development of the human brainstem PB nucleus and its related systems.
1 Beckstead, R.M., Morse, J.R. and Norgren, R., The nucleus of solitary tract in the monkey: projections to the thalamus and brainstem nuclei, J. Comp. Neuroi., 190 (1980) 259-282. 2 Berk, M.L. and Finkelstein, J.A., Efferent connections of the lateral hypothallamic area of the rat: an autoradiographic investigation, Brain Res. Bull., 8 (1982)511-526. 3 Betrand, F. and Hugelin, A., Respiratory synchronizing functions of the nucleus parabrachialis medialis: pneumotaxic mechanism, J. NeurophysioL, 34 (1971) 189-207, 4 Del Fiacco, M,, Dessi, M.L., Atzori, M.G. and Levanti, M.C., Substance P in the human brain stem. Preliminary results of its immunohistochemical localization 264 (1983) 142-147. 5 Hamilton, R.B., Ellenberger, H., Liskowski, D. and Schneiderman, N., Parabrachiai area as mediator of brachycardia in the rabbits, J. Auton. Nerv. Syst., 4 (1981) 261-281. 6 Hamilton, W.J. and Mossman, H.W., Human embryology: prena-
tal development of form and function, in: Growth of the Eml~tyo and Fetus; Dez'elopment of External Forms; Exthnation of Embty. onic and Fetal Age; 4th edn., W. Heifers, Cambridge, 1972, pp.
174-191. 7 Hsu, S.M., Raine, L. and Fanger, H., The use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase technique: a comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem, 29 (1981) 575-578. 8 Krettek, J.E. and Price, J.L., Amygdaloid projections to subcortical structures within the basal forebrain and brainstem in the rat and cat J. Comp. Neurol., 178 (1978)225-254. 9 Ljungdahl, A., H6kfelt, T. and Nilsson, G., Distribu~tion of substance P-like immunoreactivity in the central nervous system of rat--l, Cell bodies and nerve terminals, Neuroscience, 3 (1978) 681-943. 10 Loewy, A,D. and Burton, H., Nuclei of the solitary tract: efferent projections to the lower brainstem and spinal cord oT the cat, J. Comp. Neurol. 181 (1978) 421-450. I1 Loewy, A.D. and McKelar, S., Neuroanatomical basiis of central cardiovascular control, Fed. Prec., 39 (1980) 2459-2503. 12 Mizukawa, K., Me(Jeer, P.L., Tago, H., Peng, J.H., McGeer, E.G. and Kiinura, H., The cholinergic system of the human hindbrain studies by choline aceltransferase immunohistochemistry and acetylcholine histochemistry, Brain Res., 379 (1986) 39-55, 13 Mraovitch, S., Kumada, M. and Reis, D.S., Role of the nucleus parabrachialis cardiovascular regulation in cat, Brain Res., 232 (1982) 57-75.
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