Ontogeny of the calcitonin gene-related peptide in the nervous system of rat brain stem: An immunohistochemical analysis

030~522/88

~~ur~s~ien~e Vol. 26, No. 3, pp. 905-926, 1988 Printedin Great Britain

$3.00 + 0.00

Pergamon Press pit Q 1988 IBRO

ONTOGENY OF THE CALCITONIN GENE-RELATED PEPTIDE IN THE NERVOUS SYSTEM OF RAT BRAIN STEM: AN IMMUNOHISTOCHEMICAL ANALYSIS Y.

KUBOTA,*~$

S. INAGAKI,~ S. SHIMADA,$ S. M. TOHYAMA?

GIRGIS,~/ M. and S. KITO*

ZADI,/

I. MACINTYRE,]]

*Third Department of Internal Medicine, Hiroshima University School of Medicine, l-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan TDepartment of Anatomy II, Osaka University Medical School, 4-3-57 Nakanoshima, Kita-ku, Osaka 530, Japan $First Department of Anatomy, Osaka City University Medical School, l-4-54 Asahimachi, Abeno-ku, Osaka 545, Japan /iDepartment of Chemical Pathology, University of London, Royal Postgraduate Medical School, Hammersmith Hospital, Ducane Road, London W12 OHS. U.K. Abstract-The ontogeny of the calcitonin gene-related peptide in the neuron system of the rat brain stem was investigated by means of the indirect immunofluorescence technique. Calcitonin gene-related peptide-like immunoreactivity was first detected in the fibers of the nucleus of spinal tract trigeminal nerve on gestational day 18, and thereafter appeared gradually in various brain stem areas such as in the fibers of the solitary tract, gracile nucleus, cuneate nucleus, inferior colliculus, superior collicuJus, medial geniculate nucleus and in the neurons of the hypoglossal nucleus, facial nucleus, superior olive, parabrachial area, superior colliculus and peripeduncular nucleus. in colchicine-untreated animals, the immunoreactive fibers increased in number and reached adult level by postnatal day 14, whereas the number of cells reached a maximum between postnatal days 2 and 6 and then decreased in number and immunoreactivity or disappeared, except in some areas such as the superior olive and peripeduncular nucleus, which showed the same immunoreactivity as for adult animals. With colchicine treatment, calcitonin gene-related peptide-like immunoreactive cells were found in more areas of the brain stem such as the abducens nucleus, parabigeminal nucleus, principal oculomotor nucleus, trochlear nucleus and central gray, along with the nuclei which had shown calcitonin gene-related peptide immunoreactivity in the untreated animals. However, the neurons in the inferior olive showed a different ontogenetical pattern of calcitonin gene-related peptide of immunoreactivity. Immunoreactivity disappeared completely by postnatal day 21 in both colchicine-untreated and -treated animals.

The presence of the calcitonin gene-related peptide (CGRP), a peptide composed of 37 amino acids, has been recently demonstrated in nervous tissue by recombinant DNA and molecular biological techniques.‘,j4 Subsequent immunohistochemical analysis has demonstrated a wide but uneven distribution of CGRP-like immunoreactive (CGRP-LI) structures in the central and peripheral nervous systems.‘5~‘6~L7~2i~23~27.4) CGRP immunoreactivity has been found in several nerve pathways such as the hypothalamus-lateral septal area,35 ventromedial thalamic nucleus-insular cortex,j’ parabrachial area-forebrain and diencephalon,40 and the superior olivecochlea.‘* In some neurons in the central and peripheral nervous systems, CGRP co-exists with substance P 22.24choline acetyltransferase4’ or glutamate decarboxylase.“’ [‘*‘I]-CGRP binding experiments revealed a large number of binding sites in certain

areas of the central nervous system.8~ro~37 Release of CGRP from nerve terminals was found in vitro.2 Intraventricularly injected CGRP had potent effects on atria1 pressure and heart rate,6,‘9 and gastric acid secretion.3’,45 CGRP enhances muscle contraction on the endplate. 46These findings suggest that CGRP is a neuromodulator or neurotransmitter in the central and ~~pheral nervous systems. Ontogenetical studies on neuropeptides in the central nervous system4~Y~‘2~‘8~2”~‘6~38~4’~42 revealed that most peptides first appear in the rat brain from the prenatal period to postnatal week 1. The initial differentiation implies that these peptides may play an im~rtant role in brain deveiopment such as the neurotrophic factor, for some of them disappeared in adult animals. In this study we examined the ontogenetical development of the CGRP-LI neuron system in the rat brain stem by an indirect immunofluorescent method.

fTo whom correspondence

should be addressed at the Department of Anatomy II, Osaka University Medical

School, 4-3-57 Nakanoshima,

Kita-ku,

EXPERIMENTAL PROCEDURES

Osaka 530, Japan.

cholecystokinin-8; CGRP, calcitonin gene-related peptide; CGRP-LI. CGRP-like immunoreactivity; CRL, crown-rump length; PBS, phosphatebuffered saline.

Abbreviations:

CCK-8,

Experimental

animals

and preparation

of tissue (Table

I)

One male and three female animals were placed in a cage in the evening, and vaginal smears were analysed for the 905

Y. KUBOTA ef at.

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Table I. Summary of experimental animals used Number of rats used in this study

Age Gestational I6 (CRL 17 (CRL 18 (CRL 19 (CRL 20 (CRL 21 (CRL 22 (CRL

day 1415 mm) 1517mm) 17.-22mm) 22-26 mm) 2631 mm) 31-37mm) 37115 mm)

Postnatal day 0

I 2 3 4 5 6 7 8 10 I2 I4 21 28 42 56 Parentheses indicate colchicine-treated

3 4 5 4 5 5 5 10 (3) 6 (3) 8 (4) 10 (5) 6 (3) 5 (2) IO (2) 10 (4) 5 (2) 7 (2) 6 (3) 9 (5) 6 (3) g (5) 9 (6) If (15)

Immunohistochemical procedure The indirect immunofluorescence method of Coons5 was used. All sections were incubated in cold phosphate-buffered saline (PBS) for IOmin. Every third section was used for detecting CGRP. One adjacent section was used for control absorption and blocking tests, and the other adjacent section was stained with Cresyl Violet for jdentification of the brain regions. The sections for demonstration ofCGRPLI structures were incubated in CGRP antiserum diluted to I : 2000 with PBS overnight at room temperature in a humid atmosphere. The sections were rinsed 3 times for IOmin each with PBS containing 0.5% Triton X-100, followed by overnight incubation with fluorescein isothi~yanate conjugated anti-rabbit immunoglobulin G goat serum diluted at 1: 1000 with PBS. The sections were then rinsed 3 times for IOmin each with PBS and mounted in a PBS-glycerine mixture (1.1). Nomenclature Nomenclature follows that of Fulwilter and Saper,’ Jacobowitz and Palkovits,” Kiinig and Klippel,‘9 Palkovits and Jacobowitz,‘” Paxinos and Watson’” and Taber.” RESULTS Ontogeny of‘the calcitonin gene-related s_vstem in the brain stem The results are summarized

peptide neuron

in Tables 2 and 3.

rats. Gestational period

presence of sperm on the following morning; this day was designated gestational day I. Prenarul rats. Pregnant rats were anesthetized with sodium pentobarbitone (Nembutal, 20 mg/kg, i.p.) and the embryos were removed between gestational days 16 and 22. The crown-rump length (CRL) was measured to compare the dated gestational periods with the CRL dating system. Embryos with a CRL of less than IO-12 mm were immersed in Zamboni’s fixative@ and the brains were dissected in ice-cold fixative. Two types of fixation were employed for embryos with a CRL of more than 12 mm. The first group of animals was fixed as described above. The other group was perfused through the heart with ice-cold saline solution followed by Zamboni’s fixative. However, no noticeable differences between the fixation methods were found. Posrnaful ruts. Rats were examined on postnatal days O-8, IO. 12, 14, 21, 28, 42 and 56 (adult). Some of the rats were injected intraventr~cularly with colchicine (0.35 mg/kg) 12-48 h before death. All animals were killed by perfusion with Zamboni’s fixative under pentobarbital anesthesia. Preparalion of rinse. All materials were postfixed in the above fixative for 2448 h and immersed in 0. I M phosphate buffer containing 30% sucrose for 24 h. Frontal sections (IO~m) were cut serially on a cryostat. The slides were pretreated with chrom--alum gelatin to prevent detachnlent of the sections during incubation.

The characterization and specificity of antisera against CGRP were described elsewhere.‘5.‘R The antisera against CGRP were raised in rabbits against hCGRP and Tyr”-rCGRP (28-37) conjugated with ovalbumin. The specificity of these antisera was confirmed by absorption and blocking tests including: (1) replacement of the specific serum by normal rabbit serum; (2) omission of the specific serum; and (3) adsorption of each anti-CGRP sera to rat CGRP. Adsorption was carried out at 4 ‘C at the final dilution at a concentration of I x 10mhM.

Fibers Days 18and 19 ofgestation. On day 18of gestation, CGRP-LI fibers were first noted in the nucleus of the spinal tract trigeminal nerve and external cuneate nucleus. On day 19 of gestation, CGRP-LI fibers were first seen in the spinal tract of trigeminal nerve (Fig. 12A) and they appeared to be passing fibers. CGRP-LI fibers in the nucleus of spinal tract trigeminal nerve had increased in number and were ventromedially continuous to reticular formation. Days 20 and 21 qfgestation. On day 20 of gestation, CGRP-LI fibers appeared in the nucleus of solitary tract (Fig. lg, h), solitary tract (Fig. Ig), gracile nucleus (Fig. 1h) and cuneate nucleus (Fig. Ig). CGRP-LI fibers in the nucleus of spinal tract trigeminal nerve had increased and extended dorsomedially and ventromedially to the reticular formation (Fig. 1b-i). Da,v 22 of’ gestation. On day 22 of gestation, CGRP-LI fibers were first found in the external lateral, dorsal lateral and medial subdivisions of parabrachial nucleus.’ Between gestational days 20 and 22, CGRP-LI fibers in the area mentioned above increased markedly in number. Neurons CGRP-LI neurons were first detected on day 20 of gestation in the hypoglossal nucieus (Figs Ig, h, 14A) and facial nucleus (Fig. let. On day 21, CGRP-LI neurons were noted in the caudal part of the parabrachial nucleus. These CGRP-LI neurons may belong to the external lateral subnucleus of the

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Ontogeny of CGRP in rat brain stem

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parabrachial nucleus. By day 22, the CGRP-LI neurons identified in the above areas had increased in number and exhibited more intense immunofluorescence. Postnatal period Fibers Newborn ruts. The CGRP-LI fibers first appearing in the prenatal stage increased in number and those in the nucleus of spinal tract trigeminal nerve and the spinal tract trigeminal nerve had greatly increased (Fig. 2c-i). Postnatal da>js I-S. CGRP-LI fibers were found in the deep layer of superior colliculus and inferior colliculus on day 2 (Fig. IO) and in the medial geniculate nucleus, central gray and cochlear nucleus on day 4 (Fig. 1I). After postnatal day 6. CGRP-LI structures identified in the previous stages continued to increase slightly in number and density of immunofluorescence and almost reached the adult level by day 14.

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Ne~~bor~ rars. CGRP-LI cells were first seen in the inferior olive (Fig. 2h), ambiguus nucleus (Fig. 2g} and motor t~geminal nucleus (Fig. 2~). The CGRP-Ll neurons located in the hypoglossal nucleus (Fig. Zg-i), facial nucleus (Fig. 2e) and the parabrachial nucleus (Fig. 2~) had increased in number and in fluorescent intensity. Postnatal days 2-5. CGRP-LI neurons in the superior olive, superior lateral subdivision of parabrachial nucleus and the other subnucleus of the parabrachial nucleus’ (dorsal lateral and medial subnucleus), ventrolateral part of the sagulum nucleusj) and the superficial and deep layer of the superior colliculus were first noted on day 2. CGRP-LI cells located in the peri~dun~ular nucleus had increased in number and were dorsally continuous with the cell group located in the region surrounding the medial geniculate nucleus on day 2. CGRP immunorea~tivity in the neurons of the inferior olive, ambiguus and motor trigeminal nucleus disappeared on postnatal day 4. Postnatal days 6-14. CGRP-LI neurons identified in the previous stages continued to increase immunofluorescence (Figs 3, 4, 13), but some decreased in number and immunofluorescent intensity. The number of CGRP-LI neurons located in the hypoglossal nucleus reached the adult level on day 6. Then they decreased in number and immunofluorescent intensity. The cell group located in the region surrounding the medial geniculate nucleus on day 2 were restricted to the pe~pedun~ular nucleus and regions just ventral and dorsal to the medial geniculate nucleus after postnatal day 6 (Fig. 3a, b). CGRP-LI neurons on day 14 were not found in the superior colliculus and superior lateral subdivision of parabrachial nucleus (Fig. 4).

Ontogeny of CGRP in rat brain stem

After postnatal day 15. A few changes were noted in the CGRP-LI neurons in the lower brain stem after day 15. The neurons exhibited slightly stronger fluorescence than in the previous stage, but CGRP immunoreactivity in the hypoglossal nucleus disappeared on day 28 (Fig. 5). Neurons in cholchicine-treated rat Newborn rats. CGRP-LI cells were first seen in the peripeduncular nucleus (Fig. 6a) in treated animals. Postnatal days 2-5. CGRP-LI neurons detected in untreated animals showed stronger immunoreactivity and were more numerous than those in untreated animals in ~ol~hicine-treated rats on day 2. The number of CGRP-LI neurons in the inferior olive was the highest on day 2 (Fig. 1SA). The number of CGRP-LI neurons in the trigeminal motor nucleus and in the external lateral subdivisions of parabrachial nucleus reached the adult level on day 2. CGRP-LI neurons in the parabrachial nucleus were mainly in external lateral and superior lateral subdivisions. In addition, we observed a few CGRPLI neurons in other subdivisions of the parabrachial nucleus. In untreated rats, no CGRP-LI neurons were detected in the ambiguus nucleus or motor trigeminal nucleus after day 4, but they were seen in treated rats. Postnatal days 6-14. On day 6, CGRP-LI neurons were first seen in the nucleus of the solitary tract with colchicine treatment (Fig. 7h, i). CGRP-LI neurons in the inferior olive, ambiguus nucleus and motor trigetninal nucleus on day 6 (Fig. 7), and the superior colliculus and superior lateral subdivision of parabrachial nucleus on day 14 (Fig. 8), could be detected with colchicine treatment, but could not be detected in untreated rats. After pa~tnutal day 15. CGRP-LI neurons were first detected in the principal oculomotor nucleus (Fig. 9b) and trochlear nucleus (Fig. SC) on day 21. CGRP-LI neurons in the parabigeminal nucleus (Fig. 9c) and abducens nucleus (Fig. 9f) were first observed on postnatal day 28 and in the central gray on day 56 (Fig. 9b, c). On the other hand, CGRP-LI neurons in the inferior olive had disappeared by day 21 even in colchicine-treated rats (Figs 9g-j, 15B).

Aduit distribution Fibers Many CGRP-LI fibers were noted in the nucleus of spinal trigeminal nerve. A moderate number of CGRP-LI fibers were seen in the medial geniculate body, the peripheral part of the inferior colliculus, cochlear nucleus and the spinal tract of trigeminal nerve. A few were seen in the central gray, parabrachial nucleus, cuneate nucleus, gracile nucleus and nucleus of solitary tract, and very few in the superior colliculus (Figs 5, lOB, 12C).

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Neurons

CGRP-LI neurons were widely distributed in the rat brain stem. In colchicine-untreated animals, there was a moderate number of CGRP-LI neurons (about 5-20 per section) just dorsal and ventral to the medial geniculate nucleus, in the peripeduncular nucleus, external lateral nucleus of parabrachial nucleus and superior olive; a few (less than 5 per section) neurons were seen in the sagulum nucleus, ventrocaudal portion of the inferior colliculus and the nucleus of solitary tract (Fig. 5). In ~olchicine-treated rats, CGRP-LI neurons mentioned above are increased slightly. Numerous CGRP-LI neurons were found in the external lateral nucleus of parabrachial nucleus and hypoglossal nucleus. A moderate number of CGRP-LI neurons were seen in the peripeduncular nucleus, principal oculomotor nucleus, superficial and deep layer of the superior colliculus, parabigeminal nucleus, trigeminal motor nucleus, superior lateral subnucleus of parabrachial nucleus, superior olive and facial nucleus, and a few in the central gray, trochlear nucleus, sagulum nucleus, ventrocaudal portion of inferior colliculus, other subdivisions of parabrachial nucleus, abducens nucleus, ambiguus nucleus and nucleus of solitary tract (Fig. 9). DISCUSSION

Significance of colchicine treatment We used colchicine as a substrate which enhanced the immunoreactivity of cell soma. Colchicine is a drug which stopped axonal transport. Peptides were produced in the cell soma and then transported to distal regions of the neuron. With colchicine treatment only this transportational system was blocked but the ability to produce peptide remained in the neurons. The peptide produced accumulated in the cell soma. The density of the peptide in the cell soma became high, and it was then easy to detect CGRP immunoreactivity in the cell soma of CGRP producing neurons in colchicine-treated animals with immunohistochemical procedure. Calcitonin gene-relatedpeptide tures

immunoreactive struc-

The present study has demonstrated that the CGRP immunoreactivity in the rat brain stem mainly appears between the late prenatal and early neonatal periods. CGRP-LI cells could be divided into four groups from their developmental pattern. The first group appeared in the hypoglossal nucleus and facial nucleus during the prenatal period. In the second group, CGRP was first found in the perikarya in the perinatal period, in the nucleus of the solitary tract, trigeminal motor nucleus, ambiguus nucleus, superior olive, parabrachial nucleus, inferior colliculus,

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superior colliculus, sagulum nucleus and peripeduncular nucleus. The third group of CGRP-LI cells showed CGRP immunoreactivity after postnatal day 21 in the principal oculomotor nucleus, trochlear nucleus, parabigeminal nucleus and abducens nucleus. The fourth group first showed CGRP immunoreactivity in the inferior olive on postnatal day 0, and the greatest immunoreactivity was seen on day 2. Then the immunoreactivity of the neurons decreased and disappeared after day 21 in colchicinetreated and -untreated rats. CGRP-LI fibers develop unevenly from the prenatal period to postnatal day 4. In general, CGRP-LI fibers in the brain stem continue to increase in number with age and almost reach the adult level on day 14. CGRP-LI fibers in any regions of the rat brain stem did not disappear or decrease and they became the highest number in the adult. Some other peptides in the rat central nervous system disap1n contrast almost peared in the adult. 4.9.12.18.36.38.41.42 all CGRP systems in the rat brain retained immunofluorescence even in adults. This may suggest that CGRP has some important role in adult animals, such as a neurotransmitter or neuromodulator. Some considerations of the origins of calcitonin generelated peptide immunoreactivejbers natal periods

emerging in pre-

CGRP-LI fibers in the nucleus of spinal tract trigeminal nerve, spinal tract trigeminal nerve and external cuneate nucleus were first seen on postnatal day 18 or 19, when no specific CGRP-LI neurons were found in any regions of the brain stem. These fibers seem to be supplied from the trigeminal and dorsal root ganglion, and the CGRP-LI neurons in

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the dorsal root ganglion may project to the cuneate fasciculus and cuneate nucleus, which contain CGRP in adult rats.22,23 rotor

neurons

CGRP and acetylcholine are colocalized in motor neurons of the rat central nervous system,47 and CGRP enhances muscle contraction.46 CGRP first appeared in the hypoglossal nucleus, facial nucleus and motor trigeminal nucleus during the perinatal period, when suckling, using lingual, mimetic and masticatory muscles, first occurs. CGRP-LI neurons were first detected on postnatal day 21 in the principal parabrachial and trochlear nucleus of colchicinetreated rats. The CGRP in these nuclei may develop between days 15 and 21, and the CGRP-LI neurons in the abducens nucleus between days 22 and 28. The eyelids first open around day 16 in rats. Then the rat begins to use its ocular muscles innervated by the principal oculomotor, trochlear and abducens nucleus. The appearance of CGRP in motor neurons seems to correspond to the beginning of use of striated muscles. This supports the findings that CGRP enhances muscle contraction,46 and suggests that CGRP may be released from the terminals of motor neurons at neuromuscular junctions and have a role of enhancing muscle contraction, which is made with the release of acetylcholine from the same terminal. Reiationsh~p with the hypothalamic pathway

parabrach~al-ventromed~u~

Recently, fibers projecting from the superior lateral subnucleus of the parabrachial area to the ventromedial hypothalamic nucleus have been reported to

Abbreviatiom used in figures

Amb AP Cereb CG CO cu cu

D DCo

DLL DR ECU Gf 87 IC icp IO IP LC LL LVe Me5 MG ml mlf Mo5

MTz

ambiguus nucleus area postrema cerebellum central gray cochlear nucleus cuneate nucleus cuneate fasciculus nucleus of Darkschwitsch dorsal cochlear nucleus dorsal nucleus of lateral lemniscus dorsal raphe nucleus external cuneate nucleus graciie nucleus genu of facial nerve inferior colliculus inferior cerebellar inferior olive interpeduncular nucleus locus coeruleus lateral lemniscus lateral vestibular nucleus nucleus of mesencephalic tract trigeminal medial geniculate nucleus medial lemniscus medial longitudinal fasciculus motor trigeminal nucleus medial nucleus of trapezoid body

MVe PB PBe

p&z PBS Pn PP Pr5 ?

Sat3 SC Sol sol

SN

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nerve

SpVe SP5 sP5 VCO VLL 3 4 6 7 IO 12

medial

vestibular

nucleus

parabrachial nucleus parabrachial nucleus, external lateral subnucleus parabigeminal nucleus parabrachiai nucleus, superior lateral subnucleus pontine nuclei peripeduncular nucleus principal sensory trigeminal nucleus pyramidal tract red nucleus sagulum nucleus superior colliculus nucleus of solitary tract solitary tract substantia nigra superior olive spinal vestibular nucleus nucleus of spinal tract trigeminal nerve spinal tract of trigeminal nerve ventral cochlear nucleus ventral nucleus of lateral lemniscus principal oculomotor nucleus trochlear nucleus abducens nucleus facial nucleus dorsal motor nucleus of vagus hypoglossal nucleus

Ontogeny of CGRP in rat brain stem

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Fig. 1. Fig. I-9. Schematic representation of the distribution of CGRP-LI structures in frontal sections of the rat brain on gestational day 20 (E2Od) (Fig. l), untreated newborn rat (Pod) (Fig. 2), untreated rat of postnatal day 6 (P&l) (Fig. 3), untreated of postnatal day 14 fP14d) (Fig. 4), untreated of postnatal day 56 (P56d) (Fig. 5), colchicine-treated newborn rat (Fig. 6), treated of postnatal day 6 (Fig. 7). treated of postnatal day 14 (Fig. 8) and treated of postnatal day 56 (Fig. 9). The large dots indicate CGRP-LI cell groups and small dots terminal fibers. The planes are arranged from rostra1 to caudal.

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Fig. IO. Ontogeny without colchicine

of immunohistochemically stained CGRP-LI fibers in the inferior colliculus (IC) treatment. (A) Postnatal day 6. (B) Postnatal day 42. Note a remarkable increase of CGRP-LI fibers in these regions of adult rat. (A) x 225; (B) x 225.

Fig. 1I. Ontogeny of immunohistochemically stained CGRP-LI fibers in the cochlear nucleus (Co) without colchicine treatment. (A) Postnatal day 7. (B) Postnatal day 14. Note an increase of CGRP-LI fibers as the rat grows. (A) x225; (B) x 115.

Ontogeny of CGRP in rat brain stem

Fig. 12. trigs:min day 19.

ntogeny of immunohistochemically stained CGRP-LI fibers in the nucleus of spi!nal tract lerve (SpS) and spinal tract of trigeminal nerve without colchicine treatment. (A) Gc:stat Postnatal day 2. (C) Postnatal day 42. Note the remarkable increase of CGRP. .LI fibc:rs in the adult rat. (A) x 115; (B) x 115; (C) x 115.

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Fig. 13. Ontogeny of immunohistochemically stained CGRP-LI structures in the superior olive without colchicine treatment. (A) Postnatal day 7. (B) Postnatal day 14. Note an increase of CGF neurons and their imm~ofluorescent intensity as the rat grows. (A) x 1IS; (B) x 115.

Ontogeny

of CGRP

in rat brain

stem

Fig. 14. Or ltogeny of immunohistochemically stained CGRP-LI structures in the hypoglossal nuclf :us (12) days 2 (B) and 14 (C) with colchicine treatment. No te a Ima1.ked of g,esta tio nal day 20 (A), postnatal neurons as the rat grows. (A.) xl 15; 08 incrl ease Of the number and fluorescent intensity of CGRP-LI x 115; (C) x 115.

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15. Ontogeny of immunohistochemistry stained CGRP-LI structures in the inferior olive (IO) with Ihicine treatment. (A) Postnatal day 2. (B) Postnatal day 21. Note a complete disappearance of C( immunoreactivity in the neurons as the rat grows. (A) x 160; (B) x 120.

contain CGRF and cholecystokinin-8 (CCK-8),” and may be involved in the regulation of the feeding system.‘1~20~33~40 It is worth noting that the developmental patterns of CGRP and CCK-8 neurons are very similar.18 CGRP and CCK-8 neurons first appear on postnatal day 2 or 4 but disappear by day 14 in normal rats. However, colchicine treatment revealed the presence of CGRP-LI and CCK-LI cells. The distribution patterns of CGRP and CCK-8 neurons are very similar in the parabrachial area. Both types of immunoreactive fibers are concentrated in the ventrolateral portion of the ventromedial hypothalamic nucleus.“,40 These two peptides may coexist

in single cells in the superior lateral subnucleus of the parabrachial area and regulate the feeding system. Calcitonin gene-related peptide neuron system in the inferior olive

immunoreactive

In the inferior olive, CGRP-LI neurons first appeared on postnatal day 0 in colchicine-untreated and -treated rats, and increased rapidly in number and CGRP immunoreactivity, which were maximum on day 2 in treated animals. Thereafter, CGRP-LI neurons in the inferior olive decreased in number and disappeared by day 21. We previously reported that CGRP-LI fibers first appeared on day 22 of gestation

Ontogeny of CGRP in rat brain stem in developing cerebellum and increased markedly in number until postnatal days 2-4.21 These CGRP-LI fibers formed a dense plexus restricted to the Purkinje cell layer and exhibited a punctate pattern. By day 7, these CGRP-LI fibers had surrounded the Purkinje cells, but the number of fibers decreased rapidly until only a few were seen in the Purkinje cell layer. This ontogenetical pattern of CGRP-LI fibers in the cerebellum

is very similar to that of CGRP-LI

neurons

925

in the inferior olive. Thus CGRP-LI neurons are considered to send axons to the Purkinje cell layer and appear to be neurons of the olivocerebellar pathway.3 The developmental pattern of the CGRPLI neuron system in the inferior olive suggests that CGRP is related to synaptogenesis, because CGRP first appeared and increased during the period of synaptogenesis of the cerebellum’ after this period.

and disappeared

REFERENCES

Altman J. (1972) Postnatal devetopment of the cerebellar cortex in the rat II. Phases in the maturation of Purkinje

5. 6. 7. 8. 9.

10. Il. 12.

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