ontogeny of [3h]neurotensin binding sites in the rat cerebral cortex: autoradiographic study

4
Developmental Brain Research, 31 (1987) 303-306 303 Elsevier BRD 6(/193 Ontogeny of pH]neurotensin binding sites in the rat cerebral cortex: autoradiographic study Hiroshi Kiyama 1, Shinobu Inagaki 2, Shozo Kito 2 and Masaya Tohyama 3 1Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, Osaka (Japan), 2Department of Internal Medicine, Hiroshima University Medical School, Hiroshima (Japan) and 3The Second Department of Anatorny, Osaka University Medical School, Osaka (Japan) (Accepted 7 October 1986) Key words: Neurotensin binding site; Ontogeny; Autoradiography; Cerebral cortex; Rat The ontogeny of the neurotensin binding sites in the rat caudal cortex such as the retrosplenial cortex, the visual area containing area 17 and 18, the caudal somatosensory area and temporal area was examined by autoradiography. During the early postnatal peri- od (from birth to day 10), very-high-density [3H]neurotensin binding sites were observed in the caudal cortical area, but the number of binding sites decreased markedly with age. On the other hand, the ventral tegmental area and substantia nigra pars compacta had a low density of binding sites at birth, but the density increased until about day 15 and remained even in the adult rat. These findings were confirmed by the biochemical binding analysis. Neurotensin (NT) is considered to act as a neuro- transmitter or neuromodulator in the central and pe- ripheral nervous system 5-7,9-11A7-19. We found two NT systems in the rat based upon the ontogenical de- velopment of NT-like immunoreactive (NTI) struc- tures. NTI neurons in cingulate cortex, subiculum and some other regions appeared during the prenatal stage, increased markedly in number until postnatal days 7-10, and then decreased dramatically with age until only a few NTI neurons were seen. In other re- gions, NTI neurons also appeared at embryonic days 16-17, increased until the perinatal stage, and showed no decrease with age s . These findings sug- gest that NT activity in the cingulate cortex, subicu- lum and some other areas differs from that in other areas of the brain. In this study, we examined how the NT binding sites developed, and whether or not they also have tWO types of developing pattern. Animals and preparation of tissue. Male Wistar al- bino rats at postnatal days 0-1, 3-5, 14-16 and 40-60 were used. At least 3 animals at each stage were examined. All the animals were killed by deca- pitation; the brains were removed rapidly and frozen in liquid nitrogen. Serial frontal sections 10 Bm thick were cut on a cryostat, thaw-mounted on gelatin- coated slides and dried. The procedure of autoradiographic stud)'. These procedures were done according to the methods of Young and Kuhar 2°'21. Briefly, slide-mounted sec- tions were incubated in binding buffer consisting of 0.15 M Tris-HCl buffer (pH 7.6), 0.05% heat-inacti- vated bovine serum albumin and 0.1 mM bacitracin, on an ice-cold plate for 10 min. Then, the sections were incubated in buffer containing 5 nM [3HINT ([3,11-tyrosyl-3,5-3H(N)]NT spec. act. 55.3 Ci/m mol, Amersham, Japan) for 60 rain. For the control experiments the alternate sections were incubated in buffer containing both 5 nM [3H]NT and 3 ~M untri- tiated neurotensin. After the incubation, the sections were rinsed with the buffer 4x (5 min each). They were then placed on a cold plate and the tissues were dried by a stream of cold air. The dried sections were placed in apposition to tritium sensitive film (Sakura, Macroautoradiogram fihn tritium type, Sakura, Correspondence: H. Kiyama, Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, 4-3-57 Nakanoshima, Kitaku Osaka (530), Japan. 0165-3806/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

Upload: hiroshi-kiyama

Post on 20-Nov-2016

212 views

Category:

Documents


0 download

TRANSCRIPT

Developmental Brain Research, 31 (1987) 303-306 303 Elsevier

BRD 6(/193

Ontogeny of pH]neurotensin binding sites in the rat cerebral cortex: autoradiographic study

Hiroshi Kiyama 1, Shinobu Inagaki 2, Shozo Kito 2 and Masaya Tohyama 3 1Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, Osaka (Japan),

2Department of Internal Medicine, Hiroshima University Medical School, Hiroshima (Japan) and 3The Second Department of Anatorny, Osaka University Medical School, Osaka (Japan)

(Accepted 7 October 1986)

Key words: Neurotensin binding site; Ontogeny; Autoradiography; Cerebral cortex; Rat

The ontogeny of the neurotensin binding sites in the rat caudal cortex such as the retrosplenial cortex, the visual area containing area 17 and 18, the caudal somatosensory area and temporal area was examined by autoradiography. During the early postnatal peri- od (from birth to day 10), very-high-density [3H]neurotensin binding sites were observed in the caudal cortical area, but the number of binding sites decreased markedly with age. On the other hand, the ventral tegmental area and substantia nigra pars compacta had a low density of binding sites at birth, but the density increased until about day 15 and remained even in the adult rat. These findings were confirmed by the biochemical binding analysis.

Neurotensin (NT) is considered to act as a neuro-

t ransmit ter or neu romodu la to r in the central and pe- ripheral nervous system 5-7,9-11A7-19. W e found two

NT systems in the rat based upon the ontogenical de-

ve lopment of NT-like immunoreac t ive (NTI) struc-

tures. NTI neurons in cingulate cortex, subiculum

and some other regions appea red during the prenata l

stage, increased marked ly in number until postnata l

days 7 -10 , and then decreased dramatical ly with age

until only a few NTI neurons were seen. In other re-

gions, NTI neurons also appea red at embryonic days

16-17, increased until the per inata l stage, and

showed no decrease with age s . These findings sug-

gest that NT activity in the cingulate cortex, subicu-

lum and some other areas differs from that in o ther

areas of the brain. In this study, we examined how

the NT binding sites developed, and whether or not

they also have tWO types of developing pat tern.

Animals and preparation of tissue. Male Wistar al-

bino rats at postnata l days 0 - 1 , 3 -5 , 14-16 and 40-60 were used. At least 3 animals at each stage

were examined. Al l the animals were kil led by deca-

pi tat ion; the brains were removed rapidly and frozen

in liquid nitrogen. Serial frontal sections 10 Bm thick

were cut on a cryostat , thaw-mounted on gelatin-

coated slides and dried.

The procedure of autoradiographic stud)'. These

procedures were done according to the methods of

Young and Kuhar 2°'21. Briefly, s l ide-mounted sec-

tions were incubated in binding buffer consisting of

0.15 M Tris-HCl buffer (pH 7.6), 0.05% heat-inacti-

vated bovine serum albumin and 0.1 mM bacitracin,

on an ice-cold plate for 10 min. Then, the sections

were incubated in buffer containing 5 nM [3HINT

([3,11-tyrosyl-3,5-3H(N)]NT spec. act. 55.3 Ci/m

mol, Amersham, Japan) for 60 rain. For the control

exper iments the a l ternate sections were incubated in

buffer containing both 5 nM [3H]NT and 3 ~M untri-

t iated neurotensin. Af ter the incubation, the sections

were rinsed with the buffer 4 x (5 min each). They

were then placed on a cold plate and the tissues were

dried by a stream of cold air. The dried sections were

placed in apposi t ion to tritium sensitive film (Sakura,

Macroau torad iogram fihn tri t ium type, Sakura,

Correspondence: H. Kiyama, Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, 4-3-57 Nakanoshima, Kitaku Osaka (530), Japan.

0165-3806/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

304

Japan) in an X-ray cassette (Fuji, Japan), exposed for 6 weeks, and developed with Kodak Microdol X (10 min at 20 °C).

The procedure of biochemical studies. The slide- mounted sections were prepared for the hemicortex containing areas 17 and 18, or the midbrain of the brainstem containing both ventral tegmental area (VTA) and substantia nigra pars compacta (SNC). The specimens were preincubated in the binding buf- fer for 10 rain, and then in 3H-containing buffer for 60 min at 4 °C. For the detection of non-specific bind- ing, 3/~M of untritiated NT was added to the tritiated NT. Next, the sections were washed 4x (5 rain each)

at 4 °C, collected from the slide with Whatman Filter paper and placed in scintillation vials. One ml of pro- tein solubilizer (Solen 350, United Technol., Packard,

U.S.A.) was added to the vial bottles containing wiped sections and incubated for an hour at 40 °C. Before the addition of scintillation fluid (ACS II, Amersham, Japan), the vials were cooled down to room temperature. The radioactivity in the vials was counted in an Aloca scintillation counter. The pro- tein weight per section was determined according to the methods of Lowry et al. tz using bovine serum al-

bumin as a standard. Autoradiographic study. An excess of unlabeled

NT (3 ktM) in the incubation solution completely sup- pressed [3H]neurotensin binding in all sections exam- ined. Thus, the binding sites observed with [3HINT in

this study was considered to be specific.

Caudal cortex except for piriform cortical area At postnatal day 0-1, the caudal cortical areas

contained a high density of NT-binding sites, the den- sity was not uniform, the highest being in the deepest and in the most superficial layer. At days 3-5, NT binding sites in the caudal cortical area were in- creased markedly. This was most conspicuous in the retrosplenial cortex and visual area (Fig. 1A). There- after, NT-binding sites in these areas decreased gra- dually as the rats grew (cf, Fig. 1).

Other brain regions and piriform cortical area. At days 0-5, the density of NT binding sites was very low (Fig. 1A). At days 12-15, the densities in the VTA, SNC, piriform cortex surrounding the rhinai fissure, and posteromedial cortical amygdaloid nu- cleus were increased markedly (Fig. 1B) and con- tinued to increase until days 40-60 (Fig. IC).

A

B

C VTA

VTA SNC II

Fig. 1. A: autoradiogram of 5-day-old rat brain showing [3HJNT binding sites. Dense [3HINT binding sites were ob- served in the cortical area. RF: rhinal fissure. B: autoradio- gram of 15-day-old rat brain showing [3HINT binding sites. The binding sites decreased in the cortical area, though in the area surrounding RF, VTA and SNC come to have denser binding sites. C: autoradiogram of 50-day-old rat brain showing [3HINT neurotensin binding sites. Very dense binding sites were observed in the VTA and SNC.

Biochemical analysis. To confirm the autoradio- graphic results, biochemical experiments were per- formed. First, we examined whether or not the bind- ing in the neonatal rat brain was also saturable and specific. Fig. 2A showed the saturation curve of

[3HINT for the slide-mounted neonatal rat brain. The binding sites in the neonatal brain were found to be saturable and specific. Scatchard analysis showed [3H]NT binding sites of neonate with a dissociation constant (Kd) of 2.0 nM (Fig. 2B). These findings

A dpm

400

201]

100

B 2

g

\ 1 "6

v

f o - -

~ 3 5 TO [3H] Neurotensin (nM)

$ B (fmol/section)

C2000t •

o 5 1~ io AGE (postnatal day)

Fig. 2. A: saturation curve of [3H]NT binding to 1-day-old rat brain slices containing both the midbrain brainstem and cortex. The sections were incubated in various concentrations of [3H]Nq- for 60 rain. Closed circles indicate specific and closed triangles non-specific binding. [3HINT binding is expressed by dpm/section. B: Scatchard analysis of the saturation experi- ments on [3H]NT binding in l-day-old rat brain slice. The calcu- lated K d and B~n~, ~ a re : K d ~ 2.0 nM and Bm~ x = 5.0 fmol/sec- tion. C: the ontogenetic pattern of /?max values using slide- mounted sections: closed circles indicated cortex section and closed triangles indicated the midbrain section of the brainstem containing both VTA and SNC.

305

confirmed that the [3H]NT binding sites in the neona-

tal rat brain was saturable and specific,

Next, NT binding assay was performed on the cau-

dal cortex and diencephalon, because autoradiogra-

phic analysis showed quite different ontogenetical patterns for NT binding sites. The results were simi-

lar to those obtained in the autoradiographic analy-

sis. Scatchard analysis of the cortex at various stages

revealed K a of 2.15 + 0.84 nM throughout all postna-

tal stages and maximal number of binding sites (Bin,x) was 725 fmol/mg protein on day 0, 2010 fmol/mg pro- tein on day 5,188 fmol/mg protein on day' 15 and 81.1

fmol/mg protein on day 50 (Fig. 2C). On the other

hand, the Scatchard analysis of the midbrain of the

brainstem at various stages revealed K a of 2.32 +_

0.23 nM through all stages and Bma x was 85.7 fmol/mg protein on day 0, 154 fmol/mg protein on

day 5, 273 fmol/mg protein on day 15 and 227 fmol/mg protein on day 50 (Fig. 2C).

The present study has shown that the developmen-

tal pattern of NT binding sites was very different be-

tween caudal cortical areas (except for the piriform

cortical area) and other brain regions such as the VTA, SNC and area surrounding the final fissure.

The K d for the cortex and midbrain of brainstem con-

taining VTA and SNC were nearly equal (about 2.15 + 0.84 nM vs 2.32 +_ 0.23 nM) and the values did not

change with ontogenical development, although

marked differences were seen in Bma x. The Bin, x for the cortex was very high during early postnatal peri- ods, but decreased thereafter. The number of neuro-

tensin binding sites in the cortex at day 5 was 10x higher than that at day 50. The ontogenical changes

in the NT binding sites in the VTA, SNC and piriform cortical area were very similar to those of other

neurotransmitters of neuromodulators such as aden- osine, dopamine, opiates, 7-aminobutyric acid, high-

affinity muscarinic and cholinergic substances, ben- zodiazepines, and nitrendipin in the brain I 4,]3-1(~.

These binding sites increased after birth, reached the maximum number at days 10- 15 and remained even

in adult rats. This suggests that the NT and other bioactive substances mentioned above act as a neurotransmitter or neuromodulator in-the adult rat. On the other hand, the number of NT binding sites it, the caudal cortical area increased markedly from birth to day 5, after which it decreased dramatically. The reason why the level of binding site increased

306

transientally was still obscure, but the expression of

binding sites on neurons in caudal cortex might be

lost during development, or the accessibility of the

radioligand to receptor sites might change as changes

in the chemical parameter of the cortical devel-

opment occur.

1 Braestrup, C. and Nielsen, M., Ontogenetic development of benzodiazepine receptors in the rat brain, Brain Res., 147 (1978) 170-173.

2 Coyle, J.T. and Enna, S.T., Neurochemical aspects of the ontogenesis of GABAergic neurons in the rat brain, Brain Res., 111 (1976) 119-133.

3 Coyle, J.T. and Pert, C.B., Ontogenetic development of 3H-naloxone binding in rat brain, Neuropharmacology, 15 (1976) 555-560.

4 Coyle, J.T. and Yamamura, H.I., Neurochemical aspects of ontogenesis of cholinergic neurons in the rat brain, Brain Res., 118 (1976) 429-440.

5 Goedert, M., Neurotensin - - a status report, TINS, 8 (1984) 3-5.

6 Goedert, M., Lightman, S.L., Mantyh, P.W., Hunt, S.P. and Emson, P.C., Neurotensin-like immunoreactivity and neurotensin receptors in rat hypothalamus and in the inter- neuromediate lobe of the pituitary gland, Brain Res., 358 (1985) 59-69.

7 Goedert, M., Pittaway, K., Williams, B.J. and Emson, P.C., Specific binding of tritiated neurotensin to rat brain membranes: characterization and regional distribution, Brain Res., 304 (1984) 71-81.

8 Hara, Y., Shiosaka, S., Senba, E., Sakanaka, M., Inagaki, S., Takagi, H., Kawai, Y., Takatsuki, K., Matsuzaki, T. and Masaya, T., Ontogeny of the neurotensin containing neuron system of the rat: immunohistochemical analysis. I. Forebrain and diencephalon, J. Comp. Neurol., 208 (1982) 117-195.

9 Kitabgi, P., Carraway, R., Rietschoten, J.V., Granier, C., Morgat, J.L., Menez, A., Leeman, S. and Freychet, P., Neurotensin: specific binding to synaptic membranes from rat brain, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 1846-1850.

10 Kohler, C., Radesater, A.-C., Hall, H. and Winblad, B., Autoradiographic localization of [3H]neurotensin-binding sites in the hippocampal region of the rat and primate brain, Neuroscience, 16 (1985) 557-587,

11 Lazarus, L.H., Broun, M.R. and Perrin, M.H., Distribu- tion, localization and characteristics of neurotensin binding

sites in the rat brain, Neuropharmacology, 16 (1977) 625-629.

12 Lowry, O.H., Rosebrough, N.J.. Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275.

13 Marangos, P.J., Patel, J. and Stivers, J., Ontogeny of aden- osine binding sites in rat forebrain and cerebellum, J. Neu- rochem., 39 (1982) 267-270.

14 Matsubayashi, H., Kito, S., Itoga, E., Mizuno, K. and Miyoshi, R., Ontogenic development of the specific [3H]- nitrendipine binding sites in the rat whole brain. In S. Kito, T. Segawa, K. Kuriyama, H.I. Yamamura and R.W. Olsen (Eds.), Neurotransmitter Receptors, Plenum, New York, 1984, 193-204.

15 Nomura, Y., Oki, K. and Segawa, T., Ontogenetic devel- opment of the strial [3H]spiperone binding: regulation by sodium and guanine nucleotide in rat, J. Neurochem., 38 (1982) 902-908.

16 Predo, J.V., Creese, I,, Burt, D. and Snyder, S.H., Onto- genesis of dopamine receptor binding in the corpus striatum of the rat, Brain Res., 125 (1977) 376-382.

17 Uhl, G.R., Goodman, R.R. and Snyder, S.H., Neuroten- sin-containing cell bodies, fibers and nerve terminals in the brain stem of the rat: immunohistochemical mapping, Brain Res., 167 (1979) 77-91.

18 Uhl, G.R., Kuhar, M.J. and Snyder, S.H., Neurotensin: immunohistochemical localization in rat central nervous system, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 4059-4063.

19 Uhl, G.R., Whitehouse, P.J., Price, D.L., Tourtelottc, W.W. and Kuhar, M.J,, Parkinson's disease: depletion of substantia nigra neurotensin receptors, Brain Res., 308 (1984) 186-190.

20 Young, W.S., III. and Kuhar, M.J., Neurotensin recep- tors: autoradiographic localization in rat CNS. Eur. J. Pharmacol., 59 (1979) 161-163.

21 Young, W.S., III. and Kuhar, M.J., Neurotensin receptor localization by light microscopic autoradiography in rat brain, Brain Res,, 206 (1981) 273-285.