- Email: [email protected]
Localization of chick retinal 24,000 dalton protein (visinin)-like immunoreactivity in the rat lower brain stem
Neuroscience Vol. 14, No. 2, pp. 547-556, Printed in Great Britain
1985
030&4522/85 $3.00 + 0.00 Pergamon Press Ltd IBRO
LOCALIZATION OF CHICK RETINAL 24,000 DALTON PROTEIN (VISININ)-LIKE IMMUNOREACTIVITY IN THE RAT LOWER BRAIN STEM H.
KIYAMA,
K.
TAKAMI,
S. HATAKENAKA*,
and N.
I. NOMuRAf’,
M.
TOHYAMA
MIKI*
Department of Neuroanatomy, Institute of Higher Nervous Activity, TDepartment of Otolaryngology, Osaka University Medical School, 4-3-57 Nakanoshima, Kitaku, Osaka 530, Japan; *Department of Pharmacology, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920, Japan
Abstract-The distribution of visinin, a 24,000 dalton peptide, in the lower brain stem of the rat was examined by means of an indirect immunofluorescent method. Visinin-immunoreactive structures were found to be unevenly distributed only in the neuronal elements. The following neuronal systems were strongly labeled by the antiserum; the Purkinje cell system, mammillotegmental system, habenulointerpeduncular system, the second layer of the superior colliculus, ventral tegmental area, substantia nigra pars lateralis, area medial to the medial geniculate body, parabrachial area, dorsal and ventral nuclei of the lateral lemniscus, pontine reticular formation just medial to the trigeminal principal nucleus, superior olivary nucleus, solitarii nucleus, external layer of the inferior colliculus and spinal trigeminal nucleus. The densities of the labeled fibers in these areas paralleled those of the labeled cells. In addition, highly dense visinin-immunoreactive fiber plexuses were seen in the zona compacta of the substantia nigra, lateral portion of the interpeduncular nucleus, ventral tegmental nucleus of Gudden and vestibular nucleus.
Lowry et ~1.‘~with bovine serum albumin as a reference, or by the methods of Bradford when Tris was present.’
Recently a 24,000 dalton peptide, designated visinin, was isolated from the soluble proteins of the chicken retina.” The peptide appeared in the retina of 1Cday embryos and increased gradually up to the time of hatching. The concentration of visinin in the retina remained constant from hatching to adulthood.’ Immunohistochemical studies of the retina have demonstrated that the protein is concentrated in cones but not in the rods, although a small number of amacrine and horizontal cells were labeled.’ In the present study, we investigated the occurrence of visinin in the central nervous system. We report here its localization in the lower brain stem of the rat, its localization in the forebrain will be reported separately. EXPERIMENTAL
Preparation
PROCEDURES
PuriJication of vi&in
Partially purified visinin was prepared from soluble proteins of adult chicken retina by gel filtration and ion exchange column chromatography as previously described.’ The peptide was then purified by extracting a corresponding single band from sodium dodecylsulfate-polyacrylamide gels.14 The protein content was assayed by the method of
Please address all correspondence to: H. Kiyama, Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, 4-3-57 Nakanoshima, Kitaku, Osaka 530, Japan. Abbreviations: PBS, phosphate-buffered saline; visinin-IR, visinin-like immunoreactivity.
of the antiserum
against visinin
Antiserum was produced by injecting purified visinin emulsified with complete Freund’s adjuvant (300 pg) once a week into the foot pads and back of a New Zealand white rabbit (weight about 3 kg). The specificity of the antiserum against visinin was tested by a double immunodiffusion test,20 immunoelectrophoresis and a histochemical absorption test.8 The double immunodiffusion test was carried out on a 1% agar plate containing phosphate-buffered saline @H 7.4). Immunoelectrophoresis was performed on a 1% agar plate with barbital buffer (pH 8.6, ion strength 0.05). The samples were placed in wells and electrophoresed for an hour at 15 mA (2 mA/cm) at room temperature, following which rabbit anti-visinin serum was placed into the trough. Precipitation lines which developed after 24 h incubation at room temperature were stained with Amide Black IOB. Rabbit immunoglobulin was purified by ammonium sulfate precipitation (O-40%) and diethylaminoethylcellulose column chromatography eluted with 17.5 mM phosphate buffer (pH 6.3). Specificity was tested histochemically by absorption tests with the following control incubations: (1) replacement of the specific serum; (2) omission of the specific serum; (3) absorption of the anti-visinin serum to the following peptides; visinin, substance P, neurotensin, somatostatin, vasactive intestinal polypeptide, cholecystokinin- and enkephalins. Absorption was carried out at 4°C at the final dilution with each peptide ( 10m5M), followed by centrifugation at 100,OOOgfor 30 min. The supematant was used for the test. Immunohistochemical
procedures
Ten young male rats weighing 50-100 g were perfused intracardially with 50ml of saline followed by 300 ml of ice-cold Zamboni’s fixative,23 under sodium pentobarbital 547
H. Kiyama ef al.
548
anesthesia (40 mg/kg, i.p.). The brain was removed rapidly and immersed in the same fixative for 24 h and rinsed overnight in phosphate buffer containing 30% sucrose at 4’C. Serial frontal or sagital sections (10-20 pm) were cut on a cryostat, and attention was paid to the stereotaxic levels of the brain.‘5~‘s~2’The sections were immediately incubated in ice-cold phosphate-buffered saline (PBS) for 30 min before being subjected to the indirect immunofluorescent technique.6 Serial sections were divided into three groups: one for demonstrating visinin-like immunoreactive structures, the second for absorption control experiments and the third for Nissl body staining with cresyl violet. The rabbit antiserum against visinin was diluted 1: 1000 with PBS and reacted with the first group of the sections for 24 h at 15°C in a humid atmosphere. Next, the sections were rinsed first in cold PBS for IO min, then in PBS containing 1% Triton X-100 for IOmin, and lastly in PBS for IOmin. The sections were then incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit antiserum (Miles) diluted at I:2000 with PBS for 12 h at IYC followed by the same rinsing procedures as mentioned above. The sections for the absorption test were first incubated with antiserum against visinin which is absorbed by excessive visinin or peptides, after which they were nrocessed as described above. All sections were mounted in a PBS-glycerine (I : I) mixture.
RESULTS
Specificity of the untiserum
The double immunodiffusion test and immunoelectrophoresis showed a single precipitation line against purified or partially purified visinin. The histochemical absorption tests revealed all immunostaining to be absent when: (I) the specific serum was replaced by normal rabbit serum; (2) the specific serum was omitted or (3) the anti-visinin serum was absorbed with visinin. However, incubation of the antiserum absorbed with any of the other peptides resulted in no reduction in immunostaining. The positive structures identified in the present study should be described as showing visinin-like immunoreactivity, but in this paper we will use the simpler term visinin-IR. distribution of ~~sinin-like ~mmunoreQcti~~~ty in the lower brain stem Visinin-IR was exclusively localized in the neural elements. Visinin structures were seen in most parts of the lower brain stem; their distribution is represented schematically in Figs 1 and 2. cerebellum and ~est~bu~arsystem. Most Purkinje cells located in the entire cerebellum were strongly labeled by anti-visinin serum (Figs 1 and 3a), and dendritic arborizations were clearly seen as is common in Golgi staining. Furthermore, axons radiated proximally and distally from the Purkinje cells. The distal axons passed through the cerebellar medulla to the cerebellar nuclei [Figs 1 and 3bd) or vestibular nuclei (Fig. 3e) where they formed highly dense visinin-IR fiber plexuses. However, the cerebellar nuclei, vestibular nuclei and the cerebellum excluding the Purkinje cells lacked visinin-IR neurons. The
fasciculus longitudinalis medialis at the level of the nucleus raphe magnus contained a group of visininIR neurons but fibers were observed only in areas having labeled cells (Fig. 2g-i). Som~tose~sory system. The substantia gelatinosa of the dorsal horn of the spinal cord and trigeminal nucleus possessed a number of visinin-IR neurons (Fig. 2i-1). The density of the positive neurons in the trigeminal spinal nucleus varied according to the levels. The neurons were sparsely distributed at the rostra1 level, but increased in a caudal direction, the density being highest at the level of the obex (Fig. 2g-k). Visinin-IR neurons were also detected in the supratrigeminal nucleus. In all these areas, immunoreactive fibers were mainly concentrated in sites shared by labeled cells. With respect to the visual system, numerous visinin-IR cells were seen in the superior colliculus (Figs 2a-c and 4b). Most of these cells were concentrated in lamina II, but a few were scattered in laminae I and IV. The distribution of immunoreactive fibers paralleled that of immunoreactive cells. In the auditory system, nuclei of the inferior colliculus, lateral lemniscus and superior olivary contained numerous visinin-IR structures, but ail other areas were devoid of immunoreactivity (Figs 2e, g and 4d, f). In the inferior colliculus most positive cells were located in the external zone. In the lateral lemniscus, the dorsal and ventral nuclei contained numerous visinin-IR cells, Here as well. labeled fibers were mostly located in the areas adjacent to the labeled cells. Viscerosensory system. Numerous immunoreactive cells were seen in the medial portion of the nucleus tractus solitarii, commissural nucleus and lateral border of the area postrema (Figs 2i-k and Sf). A few cells were also presented in the lateral portion of the nucleus tractus solitarii. Labeled fibers were consistently seen in the vicinity of the reactive cells. Visceromotor, somutomotor and branchiomotor systerns. The nuclei associated with this system, namely
the trigeminal motor nucleus, facial nucleus, nucleus ambiguus, dorsal motor nucleus, trochlear nucleus and hypoglossai nucleus, were devoid of visinin-IR structures. Reticulur system. A few visinin-IR cells were dispersed in the midbrain, pontine and medullary reticular formation. Immunoreactive cells were concentrated in the following reticular areas (Fig. 2): the midbrain reticular formation just medial to the medial geniculate body (Figs 2a and 4a) and the area of the nucleus reticularis pontis caudalis just medial to the trigeminal principal nucleus (Figs 2f and 4e). The nucleus reticularis tegmenti pontis and nucleus reticularis also contained a small number of immunoreactive cells. The distribution pattern of the immunoreactive fibers paralleled that of the labeled cells. Ruphe system. Although raphe nuclei in general lacked immunoreactive cells, nucleus group 0, which is presumed to be a caudal continuation of the dorsal
Localization of visinin in the rat lower brain stem raphe nucleus, contained a group of immunoreactive cells (Fig. 2f). Other systems. The parabrachial area, ventral tegmental area, substantia nigra pars lateralis and the area between the lemniscus medialis and the cerebri
at the level of the decussation of the superior cerebellar peduncle, all contained a large number of visinin-IR neurons (Figs 2c-f and Sa, b). A lesser though still substantial number of immunoreactive cells were present in the central gray matter of the
Figs 1 and 2. Schematic representation of visinin-IR neurons (large dots) and fibers (small dots) in the rat cerebellum (Fie. 1) and lower brain stem (Fig. 2) on the frontal plane. Fig. 2 was arranged from rostra1 to caudal. Large dots- indicate 2&30 immunoreactive cells. x
I
,
Abbreviations
area postrema central canal nucleus cochlearis dorsalis CD colliculus inferior CI colliculus superior cs nucleus cuneiformis cu fasciculus longitudinalis medialis FLM FMTG fasciculus mammillotegmentalis fasciculus retroflexus FR G nucleus gracilis nucleus olivaris inferior IO nucleus interpeduncularis IP locus coeruleus LC lemniscus lateralis LL lemniscus medialis LM medial geniculate body MG nucleus commissuralis NC0 ND nucleus dentatus nucleus fastigius NF NI nucleus interpositus nucleus tractus mesencephali NMT nucleus tegmenti dorsalis Gudden NTD nucleus tractus solitarii NTS nucleus tegmenti ventralis Gudden NTV NV11 nucleus originis nervi facialis NVM nucleus originis nervi trigemini nucleus group 0 0 tractus corticospinalis P nucleus parabrachialis dorsalis PBD nucleus parabrachialis ventralis PBV
549
used in jigures
PCS PSN R RD RGI RL RM RPC RPOC RTP RV SG SGC SNC SNL SNR so STN SIT TB VII VL VM
vs VSP VTA X XII
pedunculus cerebellaris superior nucleus tractus spinalis nervi trigemini pars dorsomedialis nucleus ruber nucleus raphe dorsalis nucleus reticularis gigantocelbrlaris nucleus reticularis lateralis nucleus raphe magnus nucleus reticularis lateralis nucleus reticularis pontis caudalis nucleus reticularis tegmenti pontis nucleus reticularis medullae oblongatae pars ventralis substantia gelatinosa trigemini substantia grisia centralis substantia nigra zona compacta substantia nigra pars lateralis substantia nigra zona reticularis nucleus olivaris superior nucleus tractus spinalis nervi trigemini tractus spinalis nervi trigemini trapezoid body facial nerve nucleus vestibularis lateralis nucleus vestibularis medialis nucleus vestibularis superior nucleus vestibularis supinalis ventral tegmental area nucleus originis dorsalis vagi nucleus originis nervi hypoglossi
550
H. Kiyama ef al.
Fig. 2.
Fig. 3. Fluorescent photomicrographs showing ~sinin-IR~ontaining F’urkinje cell system. (a), (b) Dendritic arborization can be clearly identified. Axons from these ceils (arrow) pass through the cerebellar medulla (a, b) reaching the cerebellar nucleus (b, c, d) and vestibular nucleus (e). Frontal sections. (a), x 110; (b), x 110; (c), x 110; (d), x 150; (e), x 100. Abbreviations: ND, nucleus dentatus; NF, nucleus fastigius; NI, nucleus interpositus.
551
Fig. 4. Fluorescent photomicrographs showing visinin-containing neurons in the midbrain reticular formation just medial to the medial geniculate body (MG) (a), the second layer of the superior colliculus (b), dorsal parabrachial area (PBD) (c), dorsal nucleus of the lateral lemniscus (LL) (d), nucleus reticularis pontis caudalis just medial to the trigeminal principal nucleus (e) and superior olivary nucleus (f). Frontal sections. Abbreviations: CS, colliculus superior; LL, lemniscus lateralis; MG, medial geniculate body; PBD, nucleus parabrachialis dorsalis; SO, nucleus olivaris superior. (a)-(d), (f) x 90; (e), x 100.
552
Fig. 5. Fluorescent photomicrographs showing visinin-IR neurons in the substantia nigra pars lateralis (a), ventral tegmental area (VTA) (b) and nucleus tractus solitarii (NTS) (f), immunoreactive fiber plexus with very high density in the substantia nigra pars compacta (invading the medial portion of the pars reticulata) (b), lateral portion of the interpeduncular nucleus (IP) (d) and ventral tegmental nucleus of Gudden (NTV) (e) and visinin-IR fiber-containing fasciculus mammillotegmentalis (c). Frontal sections. Abbreviations: FMTG, fasciculus mammillotegmentalis; IP, nucleus interpeduncularis; NT!?., nucleus tractus solitarii; NTV, nucleus tegmenti ventralis Gudden; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata; VTA, ventral tegmental area. (a), x 170; (b)-(d), x 90; (e), (f), x 190.
553
Localization of visinin in the rat lower brain stem midbrain and pons (Fig. 2a-c). A few labeled cells were seen in the locus coeruleus. Immunoreactive fibers were observed, in the area mentioned above, near the labeled cells. Though highly dense visinin-IR fiber plexuses were observed, no labeled cells were found in the substantia nigra pars compacta (Figs ,?.a, b and 5b), lateral portion of the interpeduncular nucleus and ventral tegmental nucleus of Gudden (Figs 2b-e and 5d, e). In addition, the fascicilus and fasciculus retroflexus mamillotegmentalis Meynert were found to be labeled by the antisera (Figs 2a-c and SC). Fibers from these areas could be traced caudally to the ventral tegmental nucleus of Gudden and interpeduncular nucleus, and rostrally to the mammillary body and lateral portion of the medial habenular nucleus, where numerous immunoreactive cells were observed. DISCUSSION
The present study has demonstrated a wide, but uneven, distribution of visinin-IR structures in the lower brain stem. This uneven distribution indicates neurotransmitter or neuromodulator activity, or at least some function related to the systems it parallels. The neuronal systems in which visinin-IR may have a specific function were identified as the sensory system, the trigeminal descending nucleus, the auditory and visual systems, the extra pyramidal motor system, the limbic system and the autonomic system. The interpeduncular nucleus and ventral tegmental nucleus of Gudden contained a large aggregation of visinin-IR fibers. In addition, the fasciculus retroflexus Meynert and fasciculus mammillotegmental tract contained a number of visinin-IR fibers
555
which could be traced to the habenular nucleus and mammillary body where numerous visinin-IR neurons were observed. These findings suggest the visinin-IR-containing habenullopresence of interpeduncular and mammillotegmental tracts. Most Purkinje cells contained visinin-IR, with their dendritic arborization being remarkably labeled. A very high density of such fiber plexuses was observed in the cerebellar nuclei and vestibular nuclei, as well as the fiber pathways from the Purkinje cells to these nuclei. These findings indicate that visinin-IR structures are markers for Purkinje cells. The present study demonstrates a dense plexus of visinin-IR fibers in the substantia nigra pars compacta, although its origin could not be traced. Such tentative sites could be located in the substantia nigra pars lateralis, ventral tegmental area or the nucleus caudatus putamen, all containing numerous visinin-IR neurons. Visinin-IR-containing neurons in the ventral tegmental area had no observed projections to the nucleus caudatus putamen or prefrontal cortex. Thus, it is probable that visinin-IR neurons located in the ventral tegmental areas are local circuit neurons in contrast to the dopamine/cholecystokinin neurons presented in these areas.” The function of visinin-IR in the central nervous system remains obscure. In the retina of chicken embryos its function is probably related to cone In the rat brain visinin-IR first development. 9~‘o~‘9 appears at birth, which is close to the time at which the rat cerebellum begins to function.‘4,7,‘2.‘3,‘7,22 It is, therefore, likely that visinin-IR has a function related to the differentiated rather than the maturing rat cerebellum. Further studies are needed to explore the function of this new peptide.
REFERENCES
1. Addision W. H. F. (1911) The development of the Purkinje cells and of the cortical layers in the cerebellum of the rat. J. camp. Neurol. 21, 485-495. 2. Altman J. (1972) Postnatal development of the cerebellar cortex in the rat: I. The external germinal layers and the transitional molecular layer. J. camp. Neurol. 145, 353-398. 3. Altman J. (1972) Postnatal development of the cerebellar cortex in the rat: II. Phases in the maturation of Purkinje cells and of the molecular layer. J. camp. Neurol. 145, 399464. 4. Altman J. (1972) Postnatal development of the cerebellar cortex in the rat: III. Maturation of the components of the granular layer. J. camp. Neurol. 145, 465-514. 5. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteinAye binding. Analyt. Biochem. 72, 248-254. 6. Coons A. H. (1958) Fluorescent antibody methods. In General Cytochemical Methodr (ed. Danielli J. F.), pp. 39-22, Academic Press, New York. 7. Crepe1 F. (1974) Exitatory and inhibitory processes acting upon cerebellar Purkinje cells during the maturation in the rat: influence of hypothyroidism. Expl Brain Res. 20, 403-420. 8.
Grabar P. and Williams C. A. (1953) Methode pennettant l’etude conjugute des propri&&s Clectrophoretique et immunochimiquee d’un mklange de protaines. Application au s&rum sanguin. Biochim. biophys. Acta 10, 193-194.
9. Hatakenaka S., Kiyama H., Tohyama M. and Miki N. (1985) Immunohistochemical localization of chick retina 24 kd protein (visinin) in various vertebrate retina. Brain Res. In press. 10. Hatakenaka S., Kuo C. H. and Miki N. (1983) Analysis of a distinctive protein in chick retina during development. Devl Brain Res. 10, 155-163. 11. Hiikfelt T., Rehfeld J., Skirboll L., Ivemark B., Goldstain M. and Markey K. (1980) Evidence for coexistence of dopamine and CCK in the meso-limbic neurons. Nature 285, 476478. 12. Inagaki S., Sakanaka M., Shiosaka S., Senba E., Takagi H., Takatsuki K., Kawai Y., Matsuzaki T., Iida H., Hara Y. and Tohyama M. (1982) Experimental and immunohistochemical studies on the cerebellar substance P of the rat: localization, postnatal ontogeny and ways of entry to the cerebellum. Neuroscience 7, 639-645.
556
H. Kiyama et al.
13. Inagaki S., Shiosaka S., Takatsuki K., Iida H., Sakanaka M., Senba E., Hara Y., Kawai Y. and Tohyama M. (1982) Ontogeny of somatostatin containing neurons system of the rat cerebellum including its fiber connections: an experimental and immunohistochemical analysis. Devl Brain Res. 3, 500-527. 14. Laemmli IJ. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 68C-685. 15. Lame110. (1952) The mo~hogenesis and adult pattern of the lobules and fissures of the white rat. J. corny. neuron. 97, 28 1-356. 16. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275. 17. Maile I. L. and Sidman R. L. (1961) An autoradiographic
18. 19. 20. 21. 22. 23.
analysis of histogenesis in the mouse cerebellum. Expl Meurol. 4, 277-296. Meesen H. and Olsezewski J. (1949) A Cytoarchitectonic Atlas of the Rhombencephalon of the Rabbit. S. Karger, Basel. Olson M. D. (1979) Scanning electron microscopy of developing photoreceptors in the chicken retina. Anat. Rec. 193, 423-428. Ouchterlony 0. (1962) Diffusion-in-gel methods for immunological analysis. II. In Progress in Allergy (eds Kallosnd P. and Waksman B. H.). Vol. VI, pp. 30-152. Karger, Basel. Palkovits M. and Jacobowits D. M. (1974) Topographic atlas of catecholamine and acetylcholinesterase-containing neruons in the rat brain-II. Hindbrain (mesencephalon, rhombenoephalon). J. camp. Neural. 157, 2%42. Shimano T., Nosaka S. and Sasaki K. (1976) Electrophysiological study on the postnatal development of neuronal mechanisms in the rat cerebellar cortex. Brain Rex 108, 279-294. Zamboni L. and De Martin0 C. (1967) Buffered picric acid fo~aidehyde: a new rapid fixative for electron microscopy. .I. Cefl Biof. 35, 148A.
(Accepted 7 August 1984)
1985
030&4522/85 $3.00 + 0.00 Pergamon Press Ltd IBRO
LOCALIZATION OF CHICK RETINAL 24,000 DALTON PROTEIN (VISININ)-LIKE IMMUNOREACTIVITY IN THE RAT LOWER BRAIN STEM H.
KIYAMA,
K.
TAKAMI,
S. HATAKENAKA*,
and N.
I. NOMuRAf’,
M.
TOHYAMA
MIKI*
Department of Neuroanatomy, Institute of Higher Nervous Activity, TDepartment of Otolaryngology, Osaka University Medical School, 4-3-57 Nakanoshima, Kitaku, Osaka 530, Japan; *Department of Pharmacology, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920, Japan
Abstract-The distribution of visinin, a 24,000 dalton peptide, in the lower brain stem of the rat was examined by means of an indirect immunofluorescent method. Visinin-immunoreactive structures were found to be unevenly distributed only in the neuronal elements. The following neuronal systems were strongly labeled by the antiserum; the Purkinje cell system, mammillotegmental system, habenulointerpeduncular system, the second layer of the superior colliculus, ventral tegmental area, substantia nigra pars lateralis, area medial to the medial geniculate body, parabrachial area, dorsal and ventral nuclei of the lateral lemniscus, pontine reticular formation just medial to the trigeminal principal nucleus, superior olivary nucleus, solitarii nucleus, external layer of the inferior colliculus and spinal trigeminal nucleus. The densities of the labeled fibers in these areas paralleled those of the labeled cells. In addition, highly dense visinin-immunoreactive fiber plexuses were seen in the zona compacta of the substantia nigra, lateral portion of the interpeduncular nucleus, ventral tegmental nucleus of Gudden and vestibular nucleus.
Lowry et ~1.‘~with bovine serum albumin as a reference, or by the methods of Bradford when Tris was present.’
Recently a 24,000 dalton peptide, designated visinin, was isolated from the soluble proteins of the chicken retina.” The peptide appeared in the retina of 1Cday embryos and increased gradually up to the time of hatching. The concentration of visinin in the retina remained constant from hatching to adulthood.’ Immunohistochemical studies of the retina have demonstrated that the protein is concentrated in cones but not in the rods, although a small number of amacrine and horizontal cells were labeled.’ In the present study, we investigated the occurrence of visinin in the central nervous system. We report here its localization in the lower brain stem of the rat, its localization in the forebrain will be reported separately. EXPERIMENTAL
Preparation
PROCEDURES
PuriJication of vi&in
Partially purified visinin was prepared from soluble proteins of adult chicken retina by gel filtration and ion exchange column chromatography as previously described.’ The peptide was then purified by extracting a corresponding single band from sodium dodecylsulfate-polyacrylamide gels.14 The protein content was assayed by the method of
Please address all correspondence to: H. Kiyama, Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, 4-3-57 Nakanoshima, Kitaku, Osaka 530, Japan. Abbreviations: PBS, phosphate-buffered saline; visinin-IR, visinin-like immunoreactivity.
of the antiserum
against visinin
Antiserum was produced by injecting purified visinin emulsified with complete Freund’s adjuvant (300 pg) once a week into the foot pads and back of a New Zealand white rabbit (weight about 3 kg). The specificity of the antiserum against visinin was tested by a double immunodiffusion test,20 immunoelectrophoresis and a histochemical absorption test.8 The double immunodiffusion test was carried out on a 1% agar plate containing phosphate-buffered saline @H 7.4). Immunoelectrophoresis was performed on a 1% agar plate with barbital buffer (pH 8.6, ion strength 0.05). The samples were placed in wells and electrophoresed for an hour at 15 mA (2 mA/cm) at room temperature, following which rabbit anti-visinin serum was placed into the trough. Precipitation lines which developed after 24 h incubation at room temperature were stained with Amide Black IOB. Rabbit immunoglobulin was purified by ammonium sulfate precipitation (O-40%) and diethylaminoethylcellulose column chromatography eluted with 17.5 mM phosphate buffer (pH 6.3). Specificity was tested histochemically by absorption tests with the following control incubations: (1) replacement of the specific serum; (2) omission of the specific serum; (3) absorption of the anti-visinin serum to the following peptides; visinin, substance P, neurotensin, somatostatin, vasactive intestinal polypeptide, cholecystokinin- and enkephalins. Absorption was carried out at 4°C at the final dilution with each peptide ( 10m5M), followed by centrifugation at 100,OOOgfor 30 min. The supematant was used for the test. Immunohistochemical
procedures
Ten young male rats weighing 50-100 g were perfused intracardially with 50ml of saline followed by 300 ml of ice-cold Zamboni’s fixative,23 under sodium pentobarbital 547
H. Kiyama ef al.
548
anesthesia (40 mg/kg, i.p.). The brain was removed rapidly and immersed in the same fixative for 24 h and rinsed overnight in phosphate buffer containing 30% sucrose at 4’C. Serial frontal or sagital sections (10-20 pm) were cut on a cryostat, and attention was paid to the stereotaxic levels of the brain.‘5~‘s~2’The sections were immediately incubated in ice-cold phosphate-buffered saline (PBS) for 30 min before being subjected to the indirect immunofluorescent technique.6 Serial sections were divided into three groups: one for demonstrating visinin-like immunoreactive structures, the second for absorption control experiments and the third for Nissl body staining with cresyl violet. The rabbit antiserum against visinin was diluted 1: 1000 with PBS and reacted with the first group of the sections for 24 h at 15°C in a humid atmosphere. Next, the sections were rinsed first in cold PBS for IO min, then in PBS containing 1% Triton X-100 for IOmin, and lastly in PBS for IOmin. The sections were then incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit antiserum (Miles) diluted at I:2000 with PBS for 12 h at IYC followed by the same rinsing procedures as mentioned above. The sections for the absorption test were first incubated with antiserum against visinin which is absorbed by excessive visinin or peptides, after which they were nrocessed as described above. All sections were mounted in a PBS-glycerine (I : I) mixture.
RESULTS
Specificity of the untiserum
The double immunodiffusion test and immunoelectrophoresis showed a single precipitation line against purified or partially purified visinin. The histochemical absorption tests revealed all immunostaining to be absent when: (I) the specific serum was replaced by normal rabbit serum; (2) the specific serum was omitted or (3) the anti-visinin serum was absorbed with visinin. However, incubation of the antiserum absorbed with any of the other peptides resulted in no reduction in immunostaining. The positive structures identified in the present study should be described as showing visinin-like immunoreactivity, but in this paper we will use the simpler term visinin-IR. distribution of ~~sinin-like ~mmunoreQcti~~~ty in the lower brain stem Visinin-IR was exclusively localized in the neural elements. Visinin structures were seen in most parts of the lower brain stem; their distribution is represented schematically in Figs 1 and 2. cerebellum and ~est~bu~arsystem. Most Purkinje cells located in the entire cerebellum were strongly labeled by anti-visinin serum (Figs 1 and 3a), and dendritic arborizations were clearly seen as is common in Golgi staining. Furthermore, axons radiated proximally and distally from the Purkinje cells. The distal axons passed through the cerebellar medulla to the cerebellar nuclei [Figs 1 and 3bd) or vestibular nuclei (Fig. 3e) where they formed highly dense visinin-IR fiber plexuses. However, the cerebellar nuclei, vestibular nuclei and the cerebellum excluding the Purkinje cells lacked visinin-IR neurons. The
fasciculus longitudinalis medialis at the level of the nucleus raphe magnus contained a group of visininIR neurons but fibers were observed only in areas having labeled cells (Fig. 2g-i). Som~tose~sory system. The substantia gelatinosa of the dorsal horn of the spinal cord and trigeminal nucleus possessed a number of visinin-IR neurons (Fig. 2i-1). The density of the positive neurons in the trigeminal spinal nucleus varied according to the levels. The neurons were sparsely distributed at the rostra1 level, but increased in a caudal direction, the density being highest at the level of the obex (Fig. 2g-k). Visinin-IR neurons were also detected in the supratrigeminal nucleus. In all these areas, immunoreactive fibers were mainly concentrated in sites shared by labeled cells. With respect to the visual system, numerous visinin-IR cells were seen in the superior colliculus (Figs 2a-c and 4b). Most of these cells were concentrated in lamina II, but a few were scattered in laminae I and IV. The distribution of immunoreactive fibers paralleled that of immunoreactive cells. In the auditory system, nuclei of the inferior colliculus, lateral lemniscus and superior olivary contained numerous visinin-IR structures, but ail other areas were devoid of immunoreactivity (Figs 2e, g and 4d, f). In the inferior colliculus most positive cells were located in the external zone. In the lateral lemniscus, the dorsal and ventral nuclei contained numerous visinin-IR cells, Here as well. labeled fibers were mostly located in the areas adjacent to the labeled cells. Viscerosensory system. Numerous immunoreactive cells were seen in the medial portion of the nucleus tractus solitarii, commissural nucleus and lateral border of the area postrema (Figs 2i-k and Sf). A few cells were also presented in the lateral portion of the nucleus tractus solitarii. Labeled fibers were consistently seen in the vicinity of the reactive cells. Visceromotor, somutomotor and branchiomotor systerns. The nuclei associated with this system, namely
the trigeminal motor nucleus, facial nucleus, nucleus ambiguus, dorsal motor nucleus, trochlear nucleus and hypoglossai nucleus, were devoid of visinin-IR structures. Reticulur system. A few visinin-IR cells were dispersed in the midbrain, pontine and medullary reticular formation. Immunoreactive cells were concentrated in the following reticular areas (Fig. 2): the midbrain reticular formation just medial to the medial geniculate body (Figs 2a and 4a) and the area of the nucleus reticularis pontis caudalis just medial to the trigeminal principal nucleus (Figs 2f and 4e). The nucleus reticularis tegmenti pontis and nucleus reticularis also contained a small number of immunoreactive cells. The distribution pattern of the immunoreactive fibers paralleled that of the labeled cells. Ruphe system. Although raphe nuclei in general lacked immunoreactive cells, nucleus group 0, which is presumed to be a caudal continuation of the dorsal
Localization of visinin in the rat lower brain stem raphe nucleus, contained a group of immunoreactive cells (Fig. 2f). Other systems. The parabrachial area, ventral tegmental area, substantia nigra pars lateralis and the area between the lemniscus medialis and the cerebri
at the level of the decussation of the superior cerebellar peduncle, all contained a large number of visinin-IR neurons (Figs 2c-f and Sa, b). A lesser though still substantial number of immunoreactive cells were present in the central gray matter of the
Figs 1 and 2. Schematic representation of visinin-IR neurons (large dots) and fibers (small dots) in the rat cerebellum (Fie. 1) and lower brain stem (Fig. 2) on the frontal plane. Fig. 2 was arranged from rostra1 to caudal. Large dots- indicate 2&30 immunoreactive cells. x
I
,
Abbreviations
area postrema central canal nucleus cochlearis dorsalis CD colliculus inferior CI colliculus superior cs nucleus cuneiformis cu fasciculus longitudinalis medialis FLM FMTG fasciculus mammillotegmentalis fasciculus retroflexus FR G nucleus gracilis nucleus olivaris inferior IO nucleus interpeduncularis IP locus coeruleus LC lemniscus lateralis LL lemniscus medialis LM medial geniculate body MG nucleus commissuralis NC0 ND nucleus dentatus nucleus fastigius NF NI nucleus interpositus nucleus tractus mesencephali NMT nucleus tegmenti dorsalis Gudden NTD nucleus tractus solitarii NTS nucleus tegmenti ventralis Gudden NTV NV11 nucleus originis nervi facialis NVM nucleus originis nervi trigemini nucleus group 0 0 tractus corticospinalis P nucleus parabrachialis dorsalis PBD nucleus parabrachialis ventralis PBV
549
used in jigures
PCS PSN R RD RGI RL RM RPC RPOC RTP RV SG SGC SNC SNL SNR so STN SIT TB VII VL VM
vs VSP VTA X XII
pedunculus cerebellaris superior nucleus tractus spinalis nervi trigemini pars dorsomedialis nucleus ruber nucleus raphe dorsalis nucleus reticularis gigantocelbrlaris nucleus reticularis lateralis nucleus raphe magnus nucleus reticularis lateralis nucleus reticularis pontis caudalis nucleus reticularis tegmenti pontis nucleus reticularis medullae oblongatae pars ventralis substantia gelatinosa trigemini substantia grisia centralis substantia nigra zona compacta substantia nigra pars lateralis substantia nigra zona reticularis nucleus olivaris superior nucleus tractus spinalis nervi trigemini tractus spinalis nervi trigemini trapezoid body facial nerve nucleus vestibularis lateralis nucleus vestibularis medialis nucleus vestibularis superior nucleus vestibularis supinalis ventral tegmental area nucleus originis dorsalis vagi nucleus originis nervi hypoglossi
550
H. Kiyama ef al.
Fig. 2.
Fig. 3. Fluorescent photomicrographs showing ~sinin-IR~ontaining F’urkinje cell system. (a), (b) Dendritic arborization can be clearly identified. Axons from these ceils (arrow) pass through the cerebellar medulla (a, b) reaching the cerebellar nucleus (b, c, d) and vestibular nucleus (e). Frontal sections. (a), x 110; (b), x 110; (c), x 110; (d), x 150; (e), x 100. Abbreviations: ND, nucleus dentatus; NF, nucleus fastigius; NI, nucleus interpositus.
551
Fig. 4. Fluorescent photomicrographs showing visinin-containing neurons in the midbrain reticular formation just medial to the medial geniculate body (MG) (a), the second layer of the superior colliculus (b), dorsal parabrachial area (PBD) (c), dorsal nucleus of the lateral lemniscus (LL) (d), nucleus reticularis pontis caudalis just medial to the trigeminal principal nucleus (e) and superior olivary nucleus (f). Frontal sections. Abbreviations: CS, colliculus superior; LL, lemniscus lateralis; MG, medial geniculate body; PBD, nucleus parabrachialis dorsalis; SO, nucleus olivaris superior. (a)-(d), (f) x 90; (e), x 100.
552
Fig. 5. Fluorescent photomicrographs showing visinin-IR neurons in the substantia nigra pars lateralis (a), ventral tegmental area (VTA) (b) and nucleus tractus solitarii (NTS) (f), immunoreactive fiber plexus with very high density in the substantia nigra pars compacta (invading the medial portion of the pars reticulata) (b), lateral portion of the interpeduncular nucleus (IP) (d) and ventral tegmental nucleus of Gudden (NTV) (e) and visinin-IR fiber-containing fasciculus mammillotegmentalis (c). Frontal sections. Abbreviations: FMTG, fasciculus mammillotegmentalis; IP, nucleus interpeduncularis; NT!?., nucleus tractus solitarii; NTV, nucleus tegmenti ventralis Gudden; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata; VTA, ventral tegmental area. (a), x 170; (b)-(d), x 90; (e), (f), x 190.
553
Localization of visinin in the rat lower brain stem midbrain and pons (Fig. 2a-c). A few labeled cells were seen in the locus coeruleus. Immunoreactive fibers were observed, in the area mentioned above, near the labeled cells. Though highly dense visinin-IR fiber plexuses were observed, no labeled cells were found in the substantia nigra pars compacta (Figs ,?.a, b and 5b), lateral portion of the interpeduncular nucleus and ventral tegmental nucleus of Gudden (Figs 2b-e and 5d, e). In addition, the fascicilus and fasciculus retroflexus mamillotegmentalis Meynert were found to be labeled by the antisera (Figs 2a-c and SC). Fibers from these areas could be traced caudally to the ventral tegmental nucleus of Gudden and interpeduncular nucleus, and rostrally to the mammillary body and lateral portion of the medial habenular nucleus, where numerous immunoreactive cells were observed. DISCUSSION
The present study has demonstrated a wide, but uneven, distribution of visinin-IR structures in the lower brain stem. This uneven distribution indicates neurotransmitter or neuromodulator activity, or at least some function related to the systems it parallels. The neuronal systems in which visinin-IR may have a specific function were identified as the sensory system, the trigeminal descending nucleus, the auditory and visual systems, the extra pyramidal motor system, the limbic system and the autonomic system. The interpeduncular nucleus and ventral tegmental nucleus of Gudden contained a large aggregation of visinin-IR fibers. In addition, the fasciculus retroflexus Meynert and fasciculus mammillotegmental tract contained a number of visinin-IR fibers
555
which could be traced to the habenular nucleus and mammillary body where numerous visinin-IR neurons were observed. These findings suggest the visinin-IR-containing habenullopresence of interpeduncular and mammillotegmental tracts. Most Purkinje cells contained visinin-IR, with their dendritic arborization being remarkably labeled. A very high density of such fiber plexuses was observed in the cerebellar nuclei and vestibular nuclei, as well as the fiber pathways from the Purkinje cells to these nuclei. These findings indicate that visinin-IR structures are markers for Purkinje cells. The present study demonstrates a dense plexus of visinin-IR fibers in the substantia nigra pars compacta, although its origin could not be traced. Such tentative sites could be located in the substantia nigra pars lateralis, ventral tegmental area or the nucleus caudatus putamen, all containing numerous visinin-IR neurons. Visinin-IR-containing neurons in the ventral tegmental area had no observed projections to the nucleus caudatus putamen or prefrontal cortex. Thus, it is probable that visinin-IR neurons located in the ventral tegmental areas are local circuit neurons in contrast to the dopamine/cholecystokinin neurons presented in these areas.” The function of visinin-IR in the central nervous system remains obscure. In the retina of chicken embryos its function is probably related to cone In the rat brain visinin-IR first development. 9~‘o~‘9 appears at birth, which is close to the time at which the rat cerebellum begins to function.‘4,7,‘2.‘3,‘7,22 It is, therefore, likely that visinin-IR has a function related to the differentiated rather than the maturing rat cerebellum. Further studies are needed to explore the function of this new peptide.
REFERENCES
1. Addision W. H. F. (1911) The development of the Purkinje cells and of the cortical layers in the cerebellum of the rat. J. camp. Neurol. 21, 485-495. 2. Altman J. (1972) Postnatal development of the cerebellar cortex in the rat: I. The external germinal layers and the transitional molecular layer. J. camp. Neurol. 145, 353-398. 3. Altman J. (1972) Postnatal development of the cerebellar cortex in the rat: II. Phases in the maturation of Purkinje cells and of the molecular layer. J. camp. Neurol. 145, 399464. 4. Altman J. (1972) Postnatal development of the cerebellar cortex in the rat: III. Maturation of the components of the granular layer. J. camp. Neurol. 145, 465-514. 5. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteinAye binding. Analyt. Biochem. 72, 248-254. 6. Coons A. H. (1958) Fluorescent antibody methods. In General Cytochemical Methodr (ed. Danielli J. F.), pp. 39-22, Academic Press, New York. 7. Crepe1 F. (1974) Exitatory and inhibitory processes acting upon cerebellar Purkinje cells during the maturation in the rat: influence of hypothyroidism. Expl Brain Res. 20, 403-420. 8.
Grabar P. and Williams C. A. (1953) Methode pennettant l’etude conjugute des propri&&s Clectrophoretique et immunochimiquee d’un mklange de protaines. Application au s&rum sanguin. Biochim. biophys. Acta 10, 193-194.
9. Hatakenaka S., Kiyama H., Tohyama M. and Miki N. (1985) Immunohistochemical localization of chick retina 24 kd protein (visinin) in various vertebrate retina. Brain Res. In press. 10. Hatakenaka S., Kuo C. H. and Miki N. (1983) Analysis of a distinctive protein in chick retina during development. Devl Brain Res. 10, 155-163. 11. Hiikfelt T., Rehfeld J., Skirboll L., Ivemark B., Goldstain M. and Markey K. (1980) Evidence for coexistence of dopamine and CCK in the meso-limbic neurons. Nature 285, 476478. 12. Inagaki S., Sakanaka M., Shiosaka S., Senba E., Takagi H., Takatsuki K., Kawai Y., Matsuzaki T., Iida H., Hara Y. and Tohyama M. (1982) Experimental and immunohistochemical studies on the cerebellar substance P of the rat: localization, postnatal ontogeny and ways of entry to the cerebellum. Neuroscience 7, 639-645.
556
H. Kiyama et al.
13. Inagaki S., Shiosaka S., Takatsuki K., Iida H., Sakanaka M., Senba E., Hara Y., Kawai Y. and Tohyama M. (1982) Ontogeny of somatostatin containing neurons system of the rat cerebellum including its fiber connections: an experimental and immunohistochemical analysis. Devl Brain Res. 3, 500-527. 14. Laemmli IJ. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 68C-685. 15. Lame110. (1952) The mo~hogenesis and adult pattern of the lobules and fissures of the white rat. J. corny. neuron. 97, 28 1-356. 16. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275. 17. Maile I. L. and Sidman R. L. (1961) An autoradiographic
18. 19. 20. 21. 22. 23.
analysis of histogenesis in the mouse cerebellum. Expl Meurol. 4, 277-296. Meesen H. and Olsezewski J. (1949) A Cytoarchitectonic Atlas of the Rhombencephalon of the Rabbit. S. Karger, Basel. Olson M. D. (1979) Scanning electron microscopy of developing photoreceptors in the chicken retina. Anat. Rec. 193, 423-428. Ouchterlony 0. (1962) Diffusion-in-gel methods for immunological analysis. II. In Progress in Allergy (eds Kallosnd P. and Waksman B. H.). Vol. VI, pp. 30-152. Karger, Basel. Palkovits M. and Jacobowits D. M. (1974) Topographic atlas of catecholamine and acetylcholinesterase-containing neruons in the rat brain-II. Hindbrain (mesencephalon, rhombenoephalon). J. camp. Neural. 157, 2%42. Shimano T., Nosaka S. and Sasaki K. (1976) Electrophysiological study on the postnatal development of neuronal mechanisms in the rat cerebellar cortex. Brain Rex 108, 279-294. Zamboni L. and De Martin0 C. (1967) Buffered picric acid fo~aidehyde: a new rapid fixative for electron microscopy. .I. Cefl Biof. 35, 148A.
(Accepted 7 August 1984)