Evidence for the existence of three classes of neurokinin receptors in brain. Differential ontogeny of neurokinin-1, neurokinin-2 and neurokinin-3 binding sites in rat cerebral cortex

Evidence for the existence of three classes of neurokinin receptors in brain. Differential ontogeny of neurokinin-1, neurokinin-2 and neurokinin-3 binding sites in rat cerebral cortex

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Evidence for the existence of three classes of neurokinin receptors in brain. Differential ontogeny of neurokinin-1, neurokinin-2 and neurokinin-3 binding sites in rat cerebral cortex T h a n - V i n h D a m l'2, E m a n u e l Escher 2 and Rdmi Quirion l 1Douglas Hospital Research Centre and Department o[ Psychiato,, Faculty of Medicine, McGill University. Verdun. Qm~. (Canadaj and -'D~partement de Pharmacologic. Faculty;de M~decine, Universit~~de Sherbrooke, Sherbrooke, Qu& (Canada

(Accepted 15 March 1988) KQ' words: Substance P; Neurokinin A; Eledoisin: Neurokinin: Ontogeny; Cortex; Receptor

The autoradiographic distribution of the 3 neurokinin (NK) receptor sub-types, NK-1, NK-2 and NK-3, was compared in rat cerebral cortex during post-natal development using [~2Sl]Bolton-Hunter-substance P, (2-[125I]iodohistidyll)neurokinin A and [tzSl]Bolton-Hunter-eledoisin as respective radioligands. Throughout brain development, NK-1 receptor sites are present in low densities with some enrichment seen in lamina III while NK-3 binding sites are concentrated in layers IV and V. However, it appears that NK-2 receptors are mostly expressed in lamina V| and only during the first two postnatal weeks. These results demonstrate further the existence and differential ontogeny of 3 classes of NK receptors in rat brain cortex.

For many years, substance P (SP) was considered to be the only m a m m a l i a n neurokinin (NK) present in the central nervous system (CNS) 23. H o w e v e r , recent studies have revealed the existence of two o t h e r NKs, neurokinin A ( N K A ; also known as substance K) and neurokinin B ( N K B ) which are widely distributed in brain and p e r i p h e r a l tissues in various species IIA2"3°. Current research focuses on the determination of the respective physiological role of these various NK. In parallel, the existence of multiple classes of NK receptors has been p r o p o s e d on the basis of data obtained in various biosassays ~)'13'2~, and in receptor binding I-3'5')'1~ and a u t o r a d i o g r a p h i c studies 1"4"~'' lsA~,,x2,x4,27,z,~.There is now much evidence suggesting that each NK acts as preferred but not exclusive endogenous ligand for a given NK r e c e p t o r sub-type. Thus. SP, N K A and N K B preferentially binds to neurokinin-1 ( N K - I ) , neurokinin-2 (NK-2) and neurokinin-3 (NK-3) receptors, respectively ~'~'z4"2~. In the CNS, the presence of at least two NK r e c e p t o r sub-types, NK-1 and NK-3, has been clearly d e m o n -

strated L-~5"~'~::. For example, it has been shown that NK-1 binding sites are c o n c e n t r a t e d in various brain regions including the olfactory bulb, striatum, septum, dentate gyrus of the h i p p o c a m p u s , superior colliculus and locus coeruleus while NK-3 sites are mostly found in cortical areas (especially in laminae IV and V), supraoptic hypothalamic nucleus and interpeduncular nucleus 1"~'~]6-2227. H o w e v e r , conflicting results have been r e p o r t e d concerning the presence of NK-2 receptors in brain tissues. While it has been suggested that radiolabelled N K A , NKB and etedoisin ( E D ) mostly label NK-3 r e c e p t o r sites in CNS 2-4"22, we 24'25 and others aSj(' have provided some evidence for the existence of selective NK-2 receptor sites specifically labelled by N K A (but not E D and NKB) in the brain tissues. We report here additional evidence for the existence of all 3 classes of NK receptor sites in rat brain. It was found that while the respective cortical distribution of both NK- l and NK3 receptor sites remains fairly constant during postnatal ontogeny, the expression and localization of NK-2 binding sites undergoes certain modifications.

Correspondence: R. Quirion, Douglas Hospital Research Centre, 6875 LaSalle Blvd., Verdun, Que. H4H 1R3 Canada.

0006-8993/88/$03.50© 1988 Elsevier Science Publishers B.V. (Biomedical Division)

373 High densities of NK-2 sites are present in the deeper layer (lamina VI) of the cortex at postnatal days 6 and 14 but not in adult brain. This suggests the differential expression of this receptor class during brain development and demonstrates the existence of all 3 classes of NK receptors in mammalian CNS. Timed-pregnant Long-Evans female rats were obtained from Canadian Breeding Farms (St. Constant, Qu6bec). Animals were individually housed i n polyethylene cages, maintained on a 12 h light-dark cycle and given free access to food and water. After birth, pups were kept with their mothers until weaning, if applicable. Animals of either sex at various ages (6 days or 14 days postnatally, and 3 months old) were decapitated and their brains were rapidly removed from the skull and snap-frozen in 2-methylbutane at -40 °C and then kept at -80 °C until processed for autoradiography. For this purpose, brains were mounted on cryostat chucks and cut into 20/~m thick sections at -18°C. Sections were thawmounted near the edge of precleaned gelatin-coated slides, dried overnight in a desiccator at 4 °C and then stored at -80 °C until used. Frozen slidemounted brain sections were incubated for 90 min at 25 °C in a buffer containing 50 mM Tris-HCl pH 7.4, 3 mM MnC12, 0.02% (w/v) bovine serum albumin (ICN, Cleveland, OH), 2 ~g/ml chymostatin, 4 ~g/ml leupeptin, 40/zg/mi bacitracin (Sigma Chemicals, St. Louis, MO) and in the presence either 50 pM [I25I]Bolton-Hunter-SP ([125I]BH-SP) (2200 Ci/mmol) (New England Nuclear, Boston, MA) or (2-[t25I]iodohisti dyll)NKA (2000 Ci/mmol) (Amersham Canada, Ontario, Canada) or [125I]Bolton-Hunter-eledoisin ([125I]BH-ED) (2200 Ci/mmol) (New England Nuclear, Boston, MA) to study NK-1, NK-2 and NK-3 sites, respectively. Specific binding was determined as the difference in binding observed in the presence or absence of 1/~M SP, NKA or ED (Peninsula Laboratories, San Carlos, CA), respectively. At the end of the incubation, slides were placed in racks and transferred sequentially through 4 rinses (1 min each) in 50 mM Tris HCI, pH 7.4 at 4 °C. Incubated slides were rapidly dried under a stream of cold air and juxtaposed tightly against tritium-sensitive film (Ultrofilm, LKB, Sweden) and stored at room temperature as follows: 3 days for [125I]BH-SP, 15 days for (2-[ 125 I]iodohistidyl 1 )NKA and 15 days for [125I]BH-ED. After this exposure, films were deve-

loped and processed as described before 8. The comparative distribution of [125I]BH-SP, (2[125I]iodohistidyll)NKA and [125I]BH-ED binding sites in rat brain cortex during postnatal ontogeny is shown in Fig. 1. Low densities of [125I]BH-SP/NK-1 binding sites are found in various cortical areas during postnatal ontogeny (Fig. 1A,B) as well as in adult brain (Fig. 1C). Low to moderate densities of sites are observed in superficial layers throughout brain development while lamina III is enriched in NK-1 sites early after birth (P6 and P14) (Fig. 1A,B). Similar results have been reported by other groups, both during brain ontogeny26 and in adulthood 1"6'16'22'27'29. Thus, NK-1 receptor sites are present, mostly in low densities in certain cortical layers throughout postnatal brain development (Table I). The cortical expression of this receptor population does not appear to undergo major modifications during ontogeny (Table I). This is in contrast to other brain regions, such as brainstem and medulla, in which the density of sites is very high early postnatally (P1-P14) but markedly decreased thereafter to reach adult levels between the second and third week after birth 26. Similarly, the cortical distribution of [125I]BHED/NK-3 receptor sites does not appear to markedly change during brain development (Fig. 1G,H,I). Early after birth (P6), moderate to high densities of NK-3 sites are seen in layer IV (Fig. 1G), this labelling expanding to layer V by postnatal day 14 (P14) (Fig. 1H) and in adulthood (Fig. 1I). The presence of high densities of NK-3 sites in cortical laminae IV and V in adult brain has already been reported t'2'4' 6.16.22 and is confirmed here. Thus, as in the case of NK-1 receptors, NK-3 binding sites are present in certain cortical layers early postnatally, and their localization and/or expression is not markedly altered during brain ontogeny (Table I). However, the cortical distribution of (2-[~25I]iodohistidyll)NKA/putative NK-2 receptor sites undergoes major modifications during brain development (Fig. 1D,E,F and Table I). Early postnatally (P6 and P14), high densities of (2-[125I]iodohistidyll)NKA binding sites are seen mostly in lamina VI, the deeper layer of the cortex (Fig. 1D,E). This is of special interest since only low densities of NK-1 (Fig. 1A,B) and undetectable levels of NK-3 receptor sites (Fig. 1G,H) are found in this lamina during brain development. This strongly suggests that (2-[125I]iodohisti -







, IV, V

Fig. 1. Autoradiographic distribution of [12~I]BH-SP (A, B and C), (2-[125I]iodohistidyll)NKA (D, E and F) and [125I]BH-ED (G, H and I) binding sites in rat brain cortex during ontogeny (upper row, P6, 6th day postnatally; middle row, P14, 14th day postnatally; lower row, adult, 3 months old). Brain sections were incubated for 90 min at 25 °C in a buffer containing 50 mM Tris-HCl pH 7.4, 3 mM MnCI2, 0.02% (w/v) bovine serum albumin, 2 flg/ml chymostatin, 4 ktg/ml leupeptin, 40/~g/ml bacitracin and in the presence of 50 pM [125IlBH-SP, (2-[t25I]iodohistidyll)NKA or [J25I]BH-ED. Note the differential distribution of NK-1, NK-2 and NK-3 binding sites during brain ontogeny. For example, NK-I sites are mostly found in superficial layers (A, B and C) while NK-2 (D, E and F) and NK-3 (G, H and I) are localized to deep (VI) and mid-layers (IV, V), respectively. Abbreviations used: cc, corpus callosum; I to VI, cortical laminae I to VI.

375 TABLE I Laminar distribution of neurokinin (NK) receptor sub-types in rat brain cortex during post-natal development Age

Receptor sub-type NK-1



Lamina I (+) Lamina III (+ +)

Lamina VI (+ + +) Lamina III (+)

Lamina IV (+ +)

14 days

Laminae I, III (+ +) Lamina VI (+)

Lamina VI (+ + +) Lamina III (+)

Laminae IV, V (++++)

Adult (3 months)

Lamina III (+)

Very little selective binding

Laminae IV, V (+ ++ +)

6 days

dyll)NKA selectively binds to NK-2 receptor sites (and not to a significant extent to either NK-1 or NK3 receptors) in rat brain cortex during postnatal ontogeny. This also further demonstrates the existence of NK-2 receptor sites in mammalian CNS tissues 15'16' 24,25 Interestingly, (2-[125I]iodohistidyll)NKA does not bind to cortical lamina VI in adult brain tissues (Fig. IF and Table I). Thus, it would appear that the expression and appearance of NK-2 receptor sites in the deeper cortical layer (layer VI) is limited to the first few postnatal weeks being apparently shut-off later on during adulthood. It would certainly be of great interest to compare the ontogenic appearance of SP, NKA and NKB in rat cortex in order to study their correlation with the development of the three NK receptor classes. In summary, the respective laminar distribution of NK-1, NK-2 and NK-3 receptor sites is markedly different and undergoes certain modifications during brain development. While the cortical localization and expression of both NK-1 and NK-3 sites is fairly constant throughout ontogeny, the distribution and apparent expression of NK-2 receptor sites is altered with high densities of sites found in lamina VI up to 14 days postnataily, but not in adulthood. This clearly demonstrates the existence of NK-2 receptors in

the CNS. Additionally, it suggests that NK and NK receptors could be involved in brain development and maturation. Already, there is evidence suggesting that both SP and NKA promote cell and neurite outgrowth 9'1s-21. Moreover, Masu et al. 17 first re-

1 Beaujouan, J.C., Torrens, Y., Saffroy, M. and Glowinski, J., Quantitative autoradiographic analysis of the distribution of binding sites for [~25I]Bolton-Hunterderivatives of eledoisin and substance P in the rat brain, Neuroscience, 18 (1986) 857-875. 2 Bergstr6m, L., Torrens, Y., Saffroy, M., Beaujouan, J.C., Lavielle, S., Chassaing, G., Morgat, J.L., Glowinski, J. and Marquet, A., [3H]NeurokininB and t25I-Bolton-Hunter-eledoisin label identical tachykinin binding sites in the

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ported the cloning of the NK-2/substance K receptor and Hanley and Jackson 7 pointed out the striking similarity between the sequence of this receptor and the human oncogene mas. Since this oncogene has tumorigenicity, it was suggested that NK-2 receptors may be associated with mitogenic activity of NKs. Thus, NKs could be associated with mitogenic activity of NKs. Thus, NKs could act as 'growth factors' involved in brain plasticity, in addition to their better known neurotransmitter/neuromodulator activities. This possibility certainly deserves further investigation.

This research project has been sponsored by a research grant from the Scottish Rite Foundation for Schizophrenia. T.V.D. is a holder of Studentship from the Fonds pour la Formation de Chercheurs et l'Aide h la Recherche. E.E. and R.Q. are Chercheur-Boursiers of the 'Fonds de la Recherche en Sant6 de Quebec'.

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