0306-4522/91 $3.00 + 0.00 Perpmon Press plc 0 1991 IBRO
Neuroscience Vol. 45, No. 2, pp. 341-351, 1991 Printed in Great Britain
REGIONAL DEVELOPMENT OF MUSCARINIC CHOLINERGIC BINDING SITES IN THE PRENATAL RAT BRAIN M. SCHLUMPF,+~J. M. *Institute of Pharmacology,
PALACIOS,+$
R. CORT~S$and W.
LICHTENSTEIGW*
University of Zurich, Gloriastrasse 32, CH-8006 Zurich, Switzerland fSandoz AG, CH-4002 Basel, Switzerland
Abstract--The ontogeny of muscarinic choline@ binding sites was studied in rat fetal central nervous system by in oifro autoradiographic techniques using [“H]N-methyl scopolamine as ligand (1 nM). Nonspecific binding was determined after the addition of 1 PM atropine. The main findings of this study are the early appearance of muscarinic cholinergic binding sites in fetal rat central nervous system before gestational day 14, their subsequent spread in a caudofrontal direction and the rapid change of patterns within individual brain regions. Muscarinic choline@ sites are present shortly after cell birth, though the time-lag between cell generation and expression of muscarinic sites differs between neuronal cell wnulations. Hirrh _ _ _ reeentor _ densities are noted in certain brainstem nuclei that are important for early fetal and neonatal behaviors.
Switzerland) and water, under a regular light-dark cycle (lights on: 02:00-16:OO) and at constant temperature (22 f 1°C). Gestational day (CD) 1 was defined as the stage 24 h after onset of the mating period. At GD 14-GD 16, GD 18 and GD 2@-GD 22, pregnant dams were anesthetized with chloral hydrate (7OOmg/kg, s-c.) and embryonic and fetal tissues (whole bodies up to GD 16 and whole heads from GD 18 to GD 22) were frozen immediately in isopentane cooled by liquid nitrogen. Frozen tissues were sectioned on a cryostat at 10 pm, thaw-mounted on gelatine-coated glass slides and stored at -20°C until used for incubation with radioligands. Every 50 pm, tissue sections were stained with Luxol Fast Blue and Cresyl Violet for identification of structures.
In the mammalian brain a number of receptors for neurotransmitters and drugs have been found to develop early in prenatal life.12*23*2s~M~3’ Their topographical distribution and density changes markedly in relation to the developmental stage, both in experimental animals and in humans.32 This means that drug sensitivity of individual regions in the immature brain can be very different from the adult condition. Detailed information on the prenatal stage would Seem to be important especially in the case of cholinergic systems whose development may be quite significant for brain function later in life. In the trisomy 16 mouse model we have observed considerable changes in the ontogeny of muscarinic cholinergic receptors.‘g Information on the developing cholinergic system reveals an early prenatal appearance of cholineacetyltransferase (ChAT)-positive cells in the forebrain of mice and rats.‘0,18*29*33 Virtually no data are available on the ontogeny of muscarinic choline@ binding sites; published data relate primarily to postnatal development.17*21~z4 We present here the regional development of muscarinic choline@ binding sites in the prenatal rat brain as revealed by in oifro autoradiography. [3H]N-Methyl scopolamine ([3H]NMS) was used as ligand because of its high resolution and very low background activity. The same ligand had been employed for studies in the adult animal and in the human fetal and adult brain.13*32
Incubation with radioligands Ten-micrometer cryostat sections were incubated with [3mNMS (New England Nuclear), 1 nM in 170pM Tris buffer, pH 7.4, for 1 h at room temperature. The specificity was checked by coincubation of adjacent sections with [‘HINMS and atropine (1 PM). Slides were washed and air-dried and subsequently apposed to Ultrofilm (LKB) in X-omatic cassettes (Kodak). Exposure time was three to eight weeks. Sections incubated with 1 pM atropine were completely devoid of [‘HINMS binding. RRsuLrs
Spinal cord, rhomb- and mesencephalon lum
EXPERIMENTAL PROCEDURES
Animals Time-pregnant Long Evans rats were bred in our laboratory. Receptive females were mated between 16:OO and 18: 00, sperm-positive females were housed two per cage, with free access to food (NAFAG 850, Nafag, Olten, tTo whom correspondence should be addressed. Abbreviations: ChAT, cholineacetyltransferase; GD, gestational day; [-‘H]NMS, [“H]N-methyl scopolamine.
and cerebel-
While muscarinic cholinergic binding sites are already present in peripheral organs such as the heart and the gut during late embryonic period (unpublished observations), they become detectable between GD 13 and GD 14 in the central nervous system (Fig. lA, B). At that time, labeling with [31-IjNMS is weak and restricted to spinal cord. On sagittal sections of the GD 15 and GD 16 spinal cord, distinct narrow bands of rI-IlNMS labeling are present along its most dorsal and ventral aspects (Fig. 2). These muscarinic sites are associated with structures of the mantle layer at all caudorostral levels of the spinal cord. The region around the proliferating ventricular zone of the spinal canal is
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Fig. 1. Sagittal section through whole embryo at GD 13 314. (A} Cresyf Violet stained; head (h), eye (e!. placenta (p), spinal cord (SC)( x 9.6). (B) Autoradiogram; specific [‘H]NMS binding sites are present in the spinal cord. High densities of labeling appear in inner- and outermost layers of the spinal cord. Some spe&ic labeling also occurs in unid~ti~~ structures of the lower part of the body Cx f0.4).
Fig. 2. Whole body autoradio~am (~3~]NMS) of rat fetus at GD 16, sagittal section ( x 9.8). Muscarinic cholinergic binding sites in: spinal cord (SC), intestines (i), ribs (r), heart (h), lower brainstem fib), 4uadrjgem~naf piate (4), mesencephalon (ffexure! fm), d~en~pbalon (d), and ~udat~putame~ (cp).
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Fig. 3. Muscarinic cholinergic binding sites ([‘H]NMS) on sagittal sections of whole head at three different
fetal stages of the rat: (A) GD 15 (x 13); (B) GD 18 (~d~~tt~~ (x 10.1); (C) GD 22 (x9.7). Early presence of binding sites in lower brainstem and subsequent spread into diencephalon and telencephalon. Spinal cord (SC),medulla oblongata (mo), hypoglossal nucleus (XII), cerebellum (cb), motor trigeminal nucleus (tm), quadrigeminal plate (q), thalamus (th), hypothalamus (hy), caudatt+putamen (cp), neocortex (nx), and hippocampus (hi).
devoid of sites. A high degree of topographical regional relationship between muscarinic cholinergic sites and the presence of a~tyl~hoI~nestera~ was noted (unpublished observations). In the GD 15 rhombencephalon, labeling with [3H]NMS is weak and overall evenly distributed with the exception of a few more densely labeled regions in the dorsal medulla oblongata, in the area of the
developing hypoglossal, motor and spinal trigeminal nuclei (Fig. 3A). Rather low densities of PHJNMS binding sites are seen along the ventral part of the pontine and mesencephalic flexures in the direction of the diencephalon. Under the methodological conditions of the present study, muscarinic cholinergic binding sites are detectable in forebrain regions only at GD 16 (see below) (Figs 2, 3B).
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Fig. 4. Autoradiogramof muscariniccholinergicbinding sites on frontal sections of whole fetal rat heaci at GD 18 (A, x 11.7)and GD 22 (B, x 15.6).Note the high density of binding sites in the hypoglossal nucleus (XII) and in the motor trigeminal nucleus (tm). Moderate to high densities also occur in the area of the solitary tract nucleus in the dorsal medulla, in the cerebellum(cb), and in the midbrain tegmentum(mtk
Between GD 16 and GD 18, the density of 13H]NMSlabeling increases in spinal cord and lower brainstem and now develops into differentiated patterns. The layers of high densities of muscarinic sites in the dorsal and ventral parts of the spinal cord remain prominent, but more binding sites appear in between. In medulla oblongata, labeling is particularly high in hypoglossaiand trigeminal motor nuclei (Figs 3B, C, 4A). The hypoglossal cell column emergescaudally at the level of the pyramidal decussation on both sides of the central canal extending orally up to the level of the fourth ventricle.Towards the end of the gestational period the hypoglossal motor neurons have reached a high degree of differentiation. On frontal sections the nucleus reveals a distinct semicircular shape increasing steadily in size and capacity of binding rH]NMS during further development (Fig. 4B). Lateral to the cranial vagus nerve in upper medulla oblongata, the trigeminal motor nucleus is also labeled quite intensefy at this stage. During the last gestational third, from GD I8
on, higher densities of [‘H]NMS binding become
distinct in the area of the nucleus caudaiis n. trig~ini and the spinal trigeminal nerve and within an area dorsolaterally to the hypoglossal nucleus within the genera1visceral efferentcolumn at the level of the n. dorsalis motorius n. vagi and the n. tractus solitarii (Figs 3B, C, 4A, B). This area is rather smatl in diameter and rostrocaudal extension: it stretches from the level of the opening of the fourth ventricle to the rostra1 border of the h~oglossal nucleus. Muscarinic sites now occur in the pontine gray and laterally in the trigeminal nerve in the vicinity of the cerebehar peduncle. No distinct density gradients are seen within the rne~n~ph~ic te~entum during the earlier part of the last gestational third. Gradually, labeling appears to be enhanced in the ventrat and ventrolateral mesencephalon at the level of the substantia nigra (Fig. 5) and in the colliculi. Muscarinic cholinergic sites are also present in the cerebellum from GD 18 on (Figs 3B, C, 4A, B).
Muscarinic cholinergic receptors in prenatal rat brain
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Fig. 5. Muscarinic choline@ binding sites on a frontal section of the GD 20 fetal brain at the level of the caudal thalamus, also showing parts of the lower brainstem (lb). Sites are present in the pineal gland (p), in neocortex (nx) with slightly higher density in layer I and below the cortical plate and in hypoglossal nucleus (XII); low densities are present in the area of the inferior olivary complex (01).Very high densities occur in the posterior part of the thalamus (th) and high amounts of binding sites in the substantia nigra (sn). x 14.5.
Diencephalon, striatum and neocortex
Muscat-uric cholinergic sites are present in the fetal striatum by GD 16 (Fig. 2), but still virtually absent in neocortical structures and in olfactory bulb at that time. Between GD 16 and GD 18, a considerable amount of binding sites appear in the habenular nuclei, moderate amounts in the pineal, while low amounts are seen in dorsal thalamus (Figs 6, 7A). In the hypothalamus, the ontogeny of muscarinic sites does not strictly follow the pattern of cell differentiation. Cells are leaving the germinal layer of the hypothalamic third ventricle to settle in so-called tiers along a strong lateral to medial gradient with the earliest differentiating cells occupying the far-lateral positions.‘@ In the GD 18-G-D 22 hypothalamus, muscarinic sites are dense in the lateral hypothalamus (tier 1) and moderately dense in tier 2; however, high amounts are located around the IIIrd ventricle in the developing ventromedial and dorsomedial hypothalamus, in particular in the region of arcuate and ventromedial nucleus (Fig. 7A, B). The striatum represents the 6rst telencephalic location of [3H]NMS sites at GD 16 (Fig. 2). The appearance of sites in this brain region roughly parallels the major neurogenetic gradients: i.e. the sites spread from caudal to rostral, from ventral to dorsal, and from lateral to medial, with highest
densities in caudal, ventral, and lateral aspects of the structure. Faint outlining of the border of the lateral ventricle marks the presence of muscarinic sites in the basal telecephalic neuroepithelium (Fig. 8B). Muscarinic receptors in the septum appear between GD 16 and GD 18 (Fig. 8A-C) and reveal higher densities within the prospective medial septal nuclei as compared to more lateral areas.“*i4 From GD 18 on, muscarinic binding sites are also present in hippocampus (Figs 3C, 7B), approximately 24 h after they have developed in the septal region, where the septohippocampal fiber tract26 originates. From GD 16 to GD 18, neocortex exhibits a rapid unfolding. The trilaminated structure of the immature neocortex becomes discernible with the horizontally oriented cells of the subpial layer, the densely packed vertically oriented cells and the third layer of horizontal neurons above the intermediate zone.i8 [‘H]NMS sites appear in low density; they are evenly spread over the three laminae (Figs 7A, B, 8A-C). During the following gestational days, a marked increase in density is observed with a ventrolateral to dorsomedial gradient in analogy to the cortical maturation. In contrast to the precise location of benzodiazepine and beta-adrenergic receptors in the immature neocortex,28.30 the distribution of muscarinic sites in different cortical layers initially is more
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Fig. 6. Muscarinic cholinergic binding sites ([jH]NMS) at GD 16, frontal section: [jH]NMS binding at the level of the diencephalic-telencephahc junction (x 15.6). Labeling occurs mainly in the basal parts of the brain: in the caudate-putamen (cp), in the hypothalamus (hy), and to some extent in the ventrolateral frontal neocortex (nx). A high density of sites is already present in the habenular nucleus (h).
diffuse, especially in the frontal area. At late fetal stages, distinct layers are visible especially in the occipital cortex (Figs 7B, 8C). The ventrodorsal gradient of neocortical binding sites is visible at all caudorostral levels: higher densities are seen in the prospective entorhinal and pyriform cortices, in areas 2 and 3 of the temporal cortex, in the insular cortex, and in the somatosensory areas. 34On frontal sections, the thinning out of the labeling towards dorsal and mediodorsal directions (dorsal neocortex) is readily seen already at GD 18 (Figs 3B, C, 8B, C). DISCUSSION
The main findings of this study are the very early (embryonic) appearance of muscarinic cholinergic binding sites in fetal rat CNS, their progressive development in a caudofrontal direction and the rapid change of patterns within individual brain regions, a situation that distinguishes the fetal from the adult brain. Relations to neurogenesis How does the ontogeny of muscarinic cholinergic binding sites in certain areas of the fetal brain relate to regional neurogenetic events? In the rat, muscarinic cholinergic binding sites appear between GD 13 and GD 14 in spinal cord and then in lower medulla oblongata. The time of cytogenesis compares with this event insofar as peak cytogenetic events generally precede the neurochemical differentiation by about one or two days. In lower medulla, the earliest generated cells form the motor hypoglossus and
dorsal vagus nuclei with the bulk of cells being generated at GD 12.4 In upper medulla, motor nuclei are also produced over a relatively short period,5 from GD 11 to GD 13. Sensory nuclei in lower medulla, such as the nucleus gracilis, cuneatus or the nucleus of the solitary tract, are generally produced over a more prolonged time period with neurogenetic peaks at GD 13.’ In the pontine region, the trigeminal motor nucleus is an early developing cell group with peak neurogenesis at GD 12, while cells in the pontine gray are formed between GD 15 and GD 17.7 In all these brain regions, muscarinic sites are present shortly after cell birth, first in a more even distribution, then increasing in density according to regionally specific patterns. The increase is particularly prominent in the motor nuclei of the hypoglossal and trigeminal nerve between GD 16 and GD 18, in the nucleus of the solitary tract at GD 20 and, somewhat less, in the pontine gray after GD 16. In midbrain and diencephalic areas the shift in cell birth dates towards the later gestational period is mirrored in the caudorostral sequential ontogeny of muscarinic cholinergic binding sites. According to present views, major divisions of the hypothalamus and the thalamus are derived from distinguishable parts of the IIIrd ventricle epithelium.‘-4*8 Hypothalamic nuclei were assigned to four different classes according to their cytogenetic isochronicity, with the earliest arising cell groups (GD 13) in the most lateral part and subsequently arising cell groups at more medial levels.‘.2 At GD 16, muscarinic sites are present in ventrolateral hypothalamus with peak neurogenesis at GD 13, as well as in ventromedial hypothalamus where most neurons are generated
Muscarinic cholinergic receptors in prenatai rat brain
Fig. 7. [%IlNMS binding in frontal sections of the diencephalon at GD 18 (A, x 14.3) and GD 22 (B, x $3.7). Strong labeling is seen in the habeuular nuclei (h), in the area of dorso- and v~trorn~~~ h~~arn~ (hyd, hyv) and arcuate nuckus. The Gl3 18 thalamus (th) is almost devoid of binding sites, while intense labeling is seen at GD 22 in this area. Binding sites have also developed in the hippocampus (hi). Developmental gradients from v~~olatem~ to dorsomedial are seen in neocortex (nx) at both gestational days. The very faint outlining of the layers above and below the cortical plate that appears at GD 18 is not detected in neocortex at GD 22.
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Muscarinic cholinergic receptors in prenatal rat brain
between GD 13 and GD 15. Thus, the time-lag between cell generation and expression of muscarinic sites may differ between hypothalamic cell populations. In the thalamus of GD 18, muscarinic sites have developed in the region of zona incerta where the earliest cells are formed, in the lateral habenular nucleus and in the lateral geniculate nucleus composed of cells that are produced between GD 13 and GD 16.’ In the most lateral part of the habenular nucleus, muscarinic sites are first visible at GD 15, i.e. very shortly after cell generation which proceeds in a lateral to medial gradient.3 Neurons of the magnocellular basal telencephalic nuclei develop from GD 13 to GD 16 along caudorostral and lateromedial gradients. The connections between nuclei of the basal telencephalon and cortex also appear to follow a similar sequence.” The similarity in neurogenetic gradients between the various nuclei suggests that they are components of a single system spread out through the entire telencephalon.” Earlier neurons in the ventral part of the globus pallidus project to the early developing cortex near the rhinal sulcus while progressively younger neurons in the dorsal globus pallidus project to late developing dorsomedial cortical area.” These developmental processes are reflected by the evolvement of muscarinic sites which first appear in ventrolateral neocortex at GD 16 and then gradually spread to dorsomedial neocortex during the following gestational days. Neurochemical differentiation and fwtction The emergence of particular neuronal structures is intimately linked with the acquisition of certain behaviors, e.g. early motor function?’ In the human fetus, defined motor behaviors develop early in life, e.g. independent arm and leg movements at nine to 10 weeks, sucking and swallowing at 12 to 13 weeks of gestation. I5In the rat fetus, independent movement of forelimbs occurs at GD 16, of hindlimbs at GD 17 and independent opening of the mouth is seen at GD 18.9 Thus, the sequence of emergence of specific fetal behaviors bears some similarities. Little is known on neurochemical correlates of early behavioural ontogeny, but an interesting relation with muscarinic binding sites emerges. In the rat, these sites appear in medulla oblongata around GD 14, at first in quite homogeneous distribution. A more differentiated picture emerges at GD 18 when high densities of labeling are visible in the hypoglossal nucleus innervating the muscles of the tongue and in the area of trigeminal motor nucleus innervating several masticatory
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muscles. In the human fetus, a high density of muscarinic sites is observed in the hypoglossal and trigeminal nuclei at 18 weeks of gestation.‘* In both species, the appearance of muscarinic sites appears to be closely linked in time with the onset of certain functions, in support of the assumption” that motility commences as soon as structures have formed. The nuclei of the dorsally located sensory neurons arise for the most part after completion of the generation of motor neurons.5p6 At the level of the solitary tract nucleus, a sensory nucleus of the dorsal column, muscarinic binding sites are present from GD 14 on. However, the nucleus becomes strongly labeled only later in gestation, at around GD 20. The solitary tract nucleus receives afferents from the IXth and Xth cranial nerves and from the tongue. The rather sudden increase in density of muscarinic cholinergic binding sites within distinct motor or sensory nuclei of the medulla oblongata may be linked with an onset of function. Development of muscarinic cholinergic sites in concert with other neurotransmitter systems: chemical sensitivity of fetal brain structures The caudorostral gradient of appearance of muscarinic cholinergic receptor sites is also encountered with nicotinic choline& sites which become detectable in lower brainstem and spinal cord from GD 12.*’ A similar caudorostral development has been noted for benzodiazepine binding sites3’ and for the GABAergic system.“* Studies on the ontogeny of ChAT-positive cells in forebrain areas of rats also show a slight caudorostral gradient of cholinergic neurogenesis.‘s*23 Since the outgrowth of cholinergic fibers is not known in detail, the relation between the development of these projections and the ontogeny of muscarinic binding sites remains uncertain. In cerebral cortex, we observed some overlap of ChAT-positive fibers penetrating the cortex with the localization of nicotinic cholinergic receptors.23 Different ontogenetic patterns are seen, e.g. with or neurotensin25 receptors. Combeta-adrenergic,” mon to all these developmental patterns is the fact that the distribution of receptor sites in the developing brain differs greatly from the adult condition and changes rapidly in relation to the developmental stage. AS a consequence, the main target areas of a drug also differ from the adult brain and change with ongoing development. In early fetal life, the lower brainstem, where a number of receptors are seen first, can be expected to be particularly sensitive
Fig. 8. Development of muscarinic choline@ binding sites in the fetal rat striatum and frontal neocortex at GD 18 (A, x 15.6), at GD 20 (B, x 14.3), and at GD 22 (C, x 13.7). frontal sections. In caudateputamen (cp) there is a lateromedial gradient of muscarinic choline& densities at all ages. Note also the appearance of a dotted immature distribution of binding sites at GD 22 in this brain region. At GD 18 and GD 20, some muscarinic sites are present in the neuroepithelium of the basal telencephalon (bn). In neocortex (nx), a clear ventrolateral to dorsomedial gradient of binding site densities is evident. s, Septum; sb, skull bone.
M.
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to chemical influences. An example is given by certain catecholamine neuron groups which possess cholinergic receptor sites early in development23 and are responsive to nicotine during fetal life.”
Acknowledgements--We
wish to acknowledge
Itic sklllul
technical assistance of Marline Saxer and the srcretarial help of Birgit Hofer. The study was supported by rhe Swiss National Science Foundation (grant no. 3.04%0.8’)
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study of the tune of origin and settling patterns of neurons of the hypothalamus. J. camp. Neural. 182, 945-972. Altman J. and Bayer S. A. (1978) Development of the diencephalon in the rat. II. Correlation of the embryonic development of the hypothalamus with the time of origin of its neurons. J. camp. Neurol. 182, 973-994. Altman J. and Bayer S. A. (1978) Development of the diencephalon in the rat. III. Ontogeny of the specialized ventricular linings of the hypothalamic third ventricle. J. camp. Neural. 182, 995-1016. Altman J. and Bayer S. A. (1979) Development of the diencephalon in the rat. VI. Re-evaluation of the embryonic development of the thalamus on the basis of thymidine-radiographic datings. J. camp. Neurol. 188, 5Oll524. Altman J. and Bayer S. A. (1980) Development of the brain stem in the rat. I. Thymidine-radiographic study of the time of origin of neurons of the lower medulla. J. camp. Neurol. 194, l--35. Altman J. and Baver S. A. (1980) Develoument of the brain stem in the rat. II. Thvmidine-radiographic study of the time of origin of neurons of the upper medulla, excluding the vestibular and auditory nuclei. J. campy Niurol. 194; 37--56. Altman J. and Bayer S. A. (1980) Development of the brain stem in the rat. IV. Thymidine-radiographic study of the time of origin of neurons in the pontine region. J. camp. Neurol. 194, 905-929. Altman J. and Bayer S. A. (1986) The Development of the Rat Hypothalamus. Ahances in Anatomy, Embryo&y und Cell Biology, Vol. 100. Springer, Berlin. Angulo y Gonzalez A. W. (1936) The prenatal development of behavior in the albino rat. J. camp. Neurol. 55.395442. Armstrong D. M., Bruce G., Hersh L. B. and Gage F. H. (1987) Development of cholinergic neurons in the reptal/diagonal band complex of the rat. Deul Brain Res. 36, 2499256. Bayer S. A. (1985) Neurogenesis of the magnocellular basal telencephalic nuclei in the rat. Int. J. devl Neurosci. 3, 2299243.
12. Bruinink A., Lichtensteiger W. and Schlumpf M. (1983) Pre- and postnatal ontogeny and characterization of dopaminergic D2, serotonergic D2 and spirodecanone binding sites in rat forebrain. J. Neurochem. 40, 1227-1236. 13. Cortes R., Probst A., Tobler H.-J. and Palacios J. M. (1986) Muscarinic choline& receptor subtypes in the human brain. II. Quantitative autoradiographic studies. Brain Res. 362, 239-253. 14. Creps E. S. (1974) Time of neuron origin in preoptic and septal areas of the mouse: an autoradiographic study. J. camp. Neurol. 157, 161-174. 15. De Vries J. I. P., Visser G. H. A. and Prechtl H. F. R. (1984) Fetal motility in the first half of pregnancy. In Continuttq of Neural Functions from Prenatal to Postnatal Life (ed. Pmchtl H. F. R.), pp. 464%. The Lavenham Press, Lavenham. 16. Eitschberger E. (1970) Entwicklung und ChemodtQTerenzierung des Thalamus der Ratte, Advances in Anatom_v, Embryology and Cell Biology, Vol. 42, No. 6. Springer, Berlin. 17. Evans R. A., Watson M., Yamamura H. I. and Roeske W. R. (1985) Differential ontogeny of putative M, and M2 muscarinic receptor binding sites in the murine cerebral cortex and heart. J. Pharmac. exp. Ther. 235, 612618. 18. Fibiger H. C., Semba K. and Vincent S. R. (1987) Time of origin of immunohistochemically identified interneurons in the rat striatum. Sot. Neurosci. Abstr. 13, 1575. 19. Kiss J., Schlumpf M. and Balazs R. (1989) Selective retardation of the development of the basal forebrain cholinergic and pontine catecholaminergic nuclei in the brain of trisomy 16 mouse, an animal model of Down’s syndrome. Deal Brain Res. SO, 251-264.
20. Kdnig N., Roth G. and Marty R. (1975) The onset of synaptogenesis in rat temporal cortex. Anal. Embryoi., Berlin 148, 73-87. 21. Kuhar M. J., Birdsall N. J. M., Burgen A. S. V. and Hulme E. C. (1980) Ontogeny of muscarinic receptors in rat brain. Brain Res. 148, 375-383. 22. Lauder J. M., Han V. K. M., Henderson P., Veridoorn T. and Towle A. C. (1986) Prenatal ontogeny of the GABAergic system in the rat brain: an immunocytochemical study. Neuroscience 19, 465493. 23. Lichtensteiger W., Ribary U., Schlumpf M., Odermatt B. and Widmer H. R. (1988) Prenatal adverse effects of nicotine on the developing brain. Prog. Brain Res. 73, 137-157. 24. Miyoshi R., Kito S., Shimizu M. and Matsubayashi H. (1987) Ontogeny of muscarinic receptors in the rat brain with emphasis on the differentiation of M,- and M,-subtypes-semi-quantitative in vitro autoradiography. Brain Res. 420, 3022312.
25. Palacios J. M., Pazos A., Diet1 M. M., Schlumpf M. and Lichtensteiger W. (1988) The ontogeny of brain neurotensin receptors studied by autoradiography. Neuroscience 25, 307-317. 26. Powell E. W. and Hines G. (1975) Septohippocampal interface. In The Hippocampus (eds Isaacson R. L. and Probram K. H.), DD. 41-59. Plenum Press, New York. 27. Prechfl -fi. F. R. (1984) Continuity and change in early neural development. In Continuify of Neural Functionsfrom Prenatal to Postnaral Lt$e (ed. Prechtl H. F. R.), pp. 1-15. The Lavenham Press, Lavenham. 28. Ribary U. and Lichtensteiger W. (1989) Effects of acute and chronic prenatal nicotine treatment on central catecholamine systems of male and female rat fetuses and offspring. J. Pharmac. exp. Ther. 248, 786792. 29. Schambra U. B., Sulik K. K., Petrusz P. and Lauder J. M. (1989) Ontogeny of cholinergic neurons in the mouse forebrain. J. camp. Neurol. 2&I, 101-122. 30. Schlumpf M., Richard J. G., Lichtensteiger W. and Miihler H. (1933) An autoradiographic study of the prenatal development of benzodiazepine binding sites in rat brain. J. Neurosci. 3, 1478-1487. 31. Schlumpf M., Bruinink A., Lichtensteiger W., Cortes R., Palacios J. M. and Pazos A. (1987) Beta-adrenergic binding sites in fetal rat c.n.s. and nineal gland: their relation to other receptor sites. Devl Pharmac. Ther. 10,422-435. 32. Schlumpf M. and Lichtensteiger W. (1987) Benzodiazepine and muscarinic cholinergic binding sites in striatum and brainstem of the human fetus. Int. J. devl Neurosci. 5, 2833287.
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Neuroscience Vol. 45, No. 2, pp. 341-351, 1991 Printed in Great Britain
REGIONAL DEVELOPMENT OF MUSCARINIC CHOLINERGIC BINDING SITES IN THE PRENATAL RAT BRAIN M. SCHLUMPF,+~J. M. *Institute of Pharmacology,
PALACIOS,+$
R. CORT~S$and W.
LICHTENSTEIGW*
University of Zurich, Gloriastrasse 32, CH-8006 Zurich, Switzerland fSandoz AG, CH-4002 Basel, Switzerland
Abstract--The ontogeny of muscarinic choline@ binding sites was studied in rat fetal central nervous system by in oifro autoradiographic techniques using [“H]N-methyl scopolamine as ligand (1 nM). Nonspecific binding was determined after the addition of 1 PM atropine. The main findings of this study are the early appearance of muscarinic cholinergic binding sites in fetal rat central nervous system before gestational day 14, their subsequent spread in a caudofrontal direction and the rapid change of patterns within individual brain regions. Muscarinic choline@ sites are present shortly after cell birth, though the time-lag between cell generation and expression of muscarinic sites differs between neuronal cell wnulations. Hirrh _ _ _ reeentor _ densities are noted in certain brainstem nuclei that are important for early fetal and neonatal behaviors.
Switzerland) and water, under a regular light-dark cycle (lights on: 02:00-16:OO) and at constant temperature (22 f 1°C). Gestational day (CD) 1 was defined as the stage 24 h after onset of the mating period. At GD 14-GD 16, GD 18 and GD 2@-GD 22, pregnant dams were anesthetized with chloral hydrate (7OOmg/kg, s-c.) and embryonic and fetal tissues (whole bodies up to GD 16 and whole heads from GD 18 to GD 22) were frozen immediately in isopentane cooled by liquid nitrogen. Frozen tissues were sectioned on a cryostat at 10 pm, thaw-mounted on gelatine-coated glass slides and stored at -20°C until used for incubation with radioligands. Every 50 pm, tissue sections were stained with Luxol Fast Blue and Cresyl Violet for identification of structures.
In the mammalian brain a number of receptors for neurotransmitters and drugs have been found to develop early in prenatal life.12*23*2s~M~3’ Their topographical distribution and density changes markedly in relation to the developmental stage, both in experimental animals and in humans.32 This means that drug sensitivity of individual regions in the immature brain can be very different from the adult condition. Detailed information on the prenatal stage would Seem to be important especially in the case of cholinergic systems whose development may be quite significant for brain function later in life. In the trisomy 16 mouse model we have observed considerable changes in the ontogeny of muscarinic cholinergic receptors.‘g Information on the developing cholinergic system reveals an early prenatal appearance of cholineacetyltransferase (ChAT)-positive cells in the forebrain of mice and rats.‘0,18*29*33 Virtually no data are available on the ontogeny of muscarinic choline@ binding sites; published data relate primarily to postnatal development.17*21~z4 We present here the regional development of muscarinic choline@ binding sites in the prenatal rat brain as revealed by in oifro autoradiography. [3H]N-Methyl scopolamine ([3H]NMS) was used as ligand because of its high resolution and very low background activity. The same ligand had been employed for studies in the adult animal and in the human fetal and adult brain.13*32
Incubation with radioligands Ten-micrometer cryostat sections were incubated with [3mNMS (New England Nuclear), 1 nM in 170pM Tris buffer, pH 7.4, for 1 h at room temperature. The specificity was checked by coincubation of adjacent sections with [‘HINMS and atropine (1 PM). Slides were washed and air-dried and subsequently apposed to Ultrofilm (LKB) in X-omatic cassettes (Kodak). Exposure time was three to eight weeks. Sections incubated with 1 pM atropine were completely devoid of [‘HINMS binding. RRsuLrs
Spinal cord, rhomb- and mesencephalon lum
EXPERIMENTAL PROCEDURES
Animals Time-pregnant Long Evans rats were bred in our laboratory. Receptive females were mated between 16:OO and 18: 00, sperm-positive females were housed two per cage, with free access to food (NAFAG 850, Nafag, Olten, tTo whom correspondence should be addressed. Abbreviations: ChAT, cholineacetyltransferase; GD, gestational day; [-‘H]NMS, [“H]N-methyl scopolamine.
and cerebel-
While muscarinic cholinergic binding sites are already present in peripheral organs such as the heart and the gut during late embryonic period (unpublished observations), they become detectable between GD 13 and GD 14 in the central nervous system (Fig. lA, B). At that time, labeling with [31-IjNMS is weak and restricted to spinal cord. On sagittal sections of the GD 15 and GD 16 spinal cord, distinct narrow bands of rI-IlNMS labeling are present along its most dorsal and ventral aspects (Fig. 2). These muscarinic sites are associated with structures of the mantle layer at all caudorostral levels of the spinal cord. The region around the proliferating ventricular zone of the spinal canal is
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Fig. 1. Sagittal section through whole embryo at GD 13 314. (A} Cresyf Violet stained; head (h), eye (e!. placenta (p), spinal cord (SC)( x 9.6). (B) Autoradiogram; specific [‘H]NMS binding sites are present in the spinal cord. High densities of labeling appear in inner- and outermost layers of the spinal cord. Some spe&ic labeling also occurs in unid~ti~~ structures of the lower part of the body Cx f0.4).
Fig. 2. Whole body autoradio~am (~3~]NMS) of rat fetus at GD 16, sagittal section ( x 9.8). Muscarinic cholinergic binding sites in: spinal cord (SC), intestines (i), ribs (r), heart (h), lower brainstem fib), 4uadrjgem~naf piate (4), mesencephalon (ffexure! fm), d~en~pbalon (d), and ~udat~putame~ (cp).
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Fig. 3. Muscarinic cholinergic binding sites ([‘H]NMS) on sagittal sections of whole head at three different
fetal stages of the rat: (A) GD 15 (x 13); (B) GD 18 (~d~~tt~~ (x 10.1); (C) GD 22 (x9.7). Early presence of binding sites in lower brainstem and subsequent spread into diencephalon and telencephalon. Spinal cord (SC),medulla oblongata (mo), hypoglossal nucleus (XII), cerebellum (cb), motor trigeminal nucleus (tm), quadrigeminal plate (q), thalamus (th), hypothalamus (hy), caudatt+putamen (cp), neocortex (nx), and hippocampus (hi).
devoid of sites. A high degree of topographical regional relationship between muscarinic cholinergic sites and the presence of a~tyl~hoI~nestera~ was noted (unpublished observations). In the GD 15 rhombencephalon, labeling with [3H]NMS is weak and overall evenly distributed with the exception of a few more densely labeled regions in the dorsal medulla oblongata, in the area of the
developing hypoglossal, motor and spinal trigeminal nuclei (Fig. 3A). Rather low densities of PHJNMS binding sites are seen along the ventral part of the pontine and mesencephalic flexures in the direction of the diencephalon. Under the methodological conditions of the present study, muscarinic cholinergic binding sites are detectable in forebrain regions only at GD 16 (see below) (Figs 2, 3B).
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Fig. 4. Autoradiogramof muscariniccholinergicbinding sites on frontal sections of whole fetal rat heaci at GD 18 (A, x 11.7)and GD 22 (B, x 15.6).Note the high density of binding sites in the hypoglossal nucleus (XII) and in the motor trigeminal nucleus (tm). Moderate to high densities also occur in the area of the solitary tract nucleus in the dorsal medulla, in the cerebellum(cb), and in the midbrain tegmentum(mtk
Between GD 16 and GD 18, the density of 13H]NMSlabeling increases in spinal cord and lower brainstem and now develops into differentiated patterns. The layers of high densities of muscarinic sites in the dorsal and ventral parts of the spinal cord remain prominent, but more binding sites appear in between. In medulla oblongata, labeling is particularly high in hypoglossaiand trigeminal motor nuclei (Figs 3B, C, 4A). The hypoglossal cell column emergescaudally at the level of the pyramidal decussation on both sides of the central canal extending orally up to the level of the fourth ventricle.Towards the end of the gestational period the hypoglossal motor neurons have reached a high degree of differentiation. On frontal sections the nucleus reveals a distinct semicircular shape increasing steadily in size and capacity of binding rH]NMS during further development (Fig. 4B). Lateral to the cranial vagus nerve in upper medulla oblongata, the trigeminal motor nucleus is also labeled quite intensefy at this stage. During the last gestational third, from GD I8
on, higher densities of [‘H]NMS binding become
distinct in the area of the nucleus caudaiis n. trig~ini and the spinal trigeminal nerve and within an area dorsolaterally to the hypoglossal nucleus within the genera1visceral efferentcolumn at the level of the n. dorsalis motorius n. vagi and the n. tractus solitarii (Figs 3B, C, 4A, B). This area is rather smatl in diameter and rostrocaudal extension: it stretches from the level of the opening of the fourth ventricle to the rostra1 border of the h~oglossal nucleus. Muscarinic sites now occur in the pontine gray and laterally in the trigeminal nerve in the vicinity of the cerebehar peduncle. No distinct density gradients are seen within the rne~n~ph~ic te~entum during the earlier part of the last gestational third. Gradually, labeling appears to be enhanced in the ventrat and ventrolateral mesencephalon at the level of the substantia nigra (Fig. 5) and in the colliculi. Muscarinic cholinergic sites are also present in the cerebellum from GD 18 on (Figs 3B, C, 4A, B).
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Fig. 5. Muscarinic choline@ binding sites on a frontal section of the GD 20 fetal brain at the level of the caudal thalamus, also showing parts of the lower brainstem (lb). Sites are present in the pineal gland (p), in neocortex (nx) with slightly higher density in layer I and below the cortical plate and in hypoglossal nucleus (XII); low densities are present in the area of the inferior olivary complex (01).Very high densities occur in the posterior part of the thalamus (th) and high amounts of binding sites in the substantia nigra (sn). x 14.5.
Diencephalon, striatum and neocortex
Muscat-uric cholinergic sites are present in the fetal striatum by GD 16 (Fig. 2), but still virtually absent in neocortical structures and in olfactory bulb at that time. Between GD 16 and GD 18, a considerable amount of binding sites appear in the habenular nuclei, moderate amounts in the pineal, while low amounts are seen in dorsal thalamus (Figs 6, 7A). In the hypothalamus, the ontogeny of muscarinic sites does not strictly follow the pattern of cell differentiation. Cells are leaving the germinal layer of the hypothalamic third ventricle to settle in so-called tiers along a strong lateral to medial gradient with the earliest differentiating cells occupying the far-lateral positions.‘@ In the GD 18-G-D 22 hypothalamus, muscarinic sites are dense in the lateral hypothalamus (tier 1) and moderately dense in tier 2; however, high amounts are located around the IIIrd ventricle in the developing ventromedial and dorsomedial hypothalamus, in particular in the region of arcuate and ventromedial nucleus (Fig. 7A, B). The striatum represents the 6rst telencephalic location of [3H]NMS sites at GD 16 (Fig. 2). The appearance of sites in this brain region roughly parallels the major neurogenetic gradients: i.e. the sites spread from caudal to rostral, from ventral to dorsal, and from lateral to medial, with highest
densities in caudal, ventral, and lateral aspects of the structure. Faint outlining of the border of the lateral ventricle marks the presence of muscarinic sites in the basal telecephalic neuroepithelium (Fig. 8B). Muscarinic receptors in the septum appear between GD 16 and GD 18 (Fig. 8A-C) and reveal higher densities within the prospective medial septal nuclei as compared to more lateral areas.“*i4 From GD 18 on, muscarinic binding sites are also present in hippocampus (Figs 3C, 7B), approximately 24 h after they have developed in the septal region, where the septohippocampal fiber tract26 originates. From GD 16 to GD 18, neocortex exhibits a rapid unfolding. The trilaminated structure of the immature neocortex becomes discernible with the horizontally oriented cells of the subpial layer, the densely packed vertically oriented cells and the third layer of horizontal neurons above the intermediate zone.i8 [‘H]NMS sites appear in low density; they are evenly spread over the three laminae (Figs 7A, B, 8A-C). During the following gestational days, a marked increase in density is observed with a ventrolateral to dorsomedial gradient in analogy to the cortical maturation. In contrast to the precise location of benzodiazepine and beta-adrenergic receptors in the immature neocortex,28.30 the distribution of muscarinic sites in different cortical layers initially is more
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Fig. 6. Muscarinic cholinergic binding sites ([jH]NMS) at GD 16, frontal section: [jH]NMS binding at the level of the diencephalic-telencephahc junction (x 15.6). Labeling occurs mainly in the basal parts of the brain: in the caudate-putamen (cp), in the hypothalamus (hy), and to some extent in the ventrolateral frontal neocortex (nx). A high density of sites is already present in the habenular nucleus (h).
diffuse, especially in the frontal area. At late fetal stages, distinct layers are visible especially in the occipital cortex (Figs 7B, 8C). The ventrodorsal gradient of neocortical binding sites is visible at all caudorostral levels: higher densities are seen in the prospective entorhinal and pyriform cortices, in areas 2 and 3 of the temporal cortex, in the insular cortex, and in the somatosensory areas. 34On frontal sections, the thinning out of the labeling towards dorsal and mediodorsal directions (dorsal neocortex) is readily seen already at GD 18 (Figs 3B, C, 8B, C). DISCUSSION
The main findings of this study are the very early (embryonic) appearance of muscarinic cholinergic binding sites in fetal rat CNS, their progressive development in a caudofrontal direction and the rapid change of patterns within individual brain regions, a situation that distinguishes the fetal from the adult brain. Relations to neurogenesis How does the ontogeny of muscarinic cholinergic binding sites in certain areas of the fetal brain relate to regional neurogenetic events? In the rat, muscarinic cholinergic binding sites appear between GD 13 and GD 14 in spinal cord and then in lower medulla oblongata. The time of cytogenesis compares with this event insofar as peak cytogenetic events generally precede the neurochemical differentiation by about one or two days. In lower medulla, the earliest generated cells form the motor hypoglossus and
dorsal vagus nuclei with the bulk of cells being generated at GD 12.4 In upper medulla, motor nuclei are also produced over a relatively short period,5 from GD 11 to GD 13. Sensory nuclei in lower medulla, such as the nucleus gracilis, cuneatus or the nucleus of the solitary tract, are generally produced over a more prolonged time period with neurogenetic peaks at GD 13.’ In the pontine region, the trigeminal motor nucleus is an early developing cell group with peak neurogenesis at GD 12, while cells in the pontine gray are formed between GD 15 and GD 17.7 In all these brain regions, muscarinic sites are present shortly after cell birth, first in a more even distribution, then increasing in density according to regionally specific patterns. The increase is particularly prominent in the motor nuclei of the hypoglossal and trigeminal nerve between GD 16 and GD 18, in the nucleus of the solitary tract at GD 20 and, somewhat less, in the pontine gray after GD 16. In midbrain and diencephalic areas the shift in cell birth dates towards the later gestational period is mirrored in the caudorostral sequential ontogeny of muscarinic cholinergic binding sites. According to present views, major divisions of the hypothalamus and the thalamus are derived from distinguishable parts of the IIIrd ventricle epithelium.‘-4*8 Hypothalamic nuclei were assigned to four different classes according to their cytogenetic isochronicity, with the earliest arising cell groups (GD 13) in the most lateral part and subsequently arising cell groups at more medial levels.‘.2 At GD 16, muscarinic sites are present in ventrolateral hypothalamus with peak neurogenesis at GD 13, as well as in ventromedial hypothalamus where most neurons are generated
Muscarinic cholinergic receptors in prenatai rat brain
Fig. 7. [%IlNMS binding in frontal sections of the diencephalon at GD 18 (A, x 14.3) and GD 22 (B, x $3.7). Strong labeling is seen in the habeuular nuclei (h), in the area of dorso- and v~trorn~~~ h~~arn~ (hyd, hyv) and arcuate nuckus. The Gl3 18 thalamus (th) is almost devoid of binding sites, while intense labeling is seen at GD 22 in this area. Binding sites have also developed in the hippocampus (hi). Developmental gradients from v~~olatem~ to dorsomedial are seen in neocortex (nx) at both gestational days. The very faint outlining of the layers above and below the cortical plate that appears at GD 18 is not detected in neocortex at GD 22.
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Muscarinic cholinergic receptors in prenatal rat brain
between GD 13 and GD 15. Thus, the time-lag between cell generation and expression of muscarinic sites may differ between hypothalamic cell populations. In the thalamus of GD 18, muscarinic sites have developed in the region of zona incerta where the earliest cells are formed, in the lateral habenular nucleus and in the lateral geniculate nucleus composed of cells that are produced between GD 13 and GD 16.’ In the most lateral part of the habenular nucleus, muscarinic sites are first visible at GD 15, i.e. very shortly after cell generation which proceeds in a lateral to medial gradient.3 Neurons of the magnocellular basal telencephalic nuclei develop from GD 13 to GD 16 along caudorostral and lateromedial gradients. The connections between nuclei of the basal telencephalon and cortex also appear to follow a similar sequence.” The similarity in neurogenetic gradients between the various nuclei suggests that they are components of a single system spread out through the entire telencephalon.” Earlier neurons in the ventral part of the globus pallidus project to the early developing cortex near the rhinal sulcus while progressively younger neurons in the dorsal globus pallidus project to late developing dorsomedial cortical area.” These developmental processes are reflected by the evolvement of muscarinic sites which first appear in ventrolateral neocortex at GD 16 and then gradually spread to dorsomedial neocortex during the following gestational days. Neurochemical differentiation and fwtction The emergence of particular neuronal structures is intimately linked with the acquisition of certain behaviors, e.g. early motor function?’ In the human fetus, defined motor behaviors develop early in life, e.g. independent arm and leg movements at nine to 10 weeks, sucking and swallowing at 12 to 13 weeks of gestation. I5In the rat fetus, independent movement of forelimbs occurs at GD 16, of hindlimbs at GD 17 and independent opening of the mouth is seen at GD 18.9 Thus, the sequence of emergence of specific fetal behaviors bears some similarities. Little is known on neurochemical correlates of early behavioural ontogeny, but an interesting relation with muscarinic binding sites emerges. In the rat, these sites appear in medulla oblongata around GD 14, at first in quite homogeneous distribution. A more differentiated picture emerges at GD 18 when high densities of labeling are visible in the hypoglossal nucleus innervating the muscles of the tongue and in the area of trigeminal motor nucleus innervating several masticatory
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muscles. In the human fetus, a high density of muscarinic sites is observed in the hypoglossal and trigeminal nuclei at 18 weeks of gestation.‘* In both species, the appearance of muscarinic sites appears to be closely linked in time with the onset of certain functions, in support of the assumption” that motility commences as soon as structures have formed. The nuclei of the dorsally located sensory neurons arise for the most part after completion of the generation of motor neurons.5p6 At the level of the solitary tract nucleus, a sensory nucleus of the dorsal column, muscarinic binding sites are present from GD 14 on. However, the nucleus becomes strongly labeled only later in gestation, at around GD 20. The solitary tract nucleus receives afferents from the IXth and Xth cranial nerves and from the tongue. The rather sudden increase in density of muscarinic cholinergic binding sites within distinct motor or sensory nuclei of the medulla oblongata may be linked with an onset of function. Development of muscarinic cholinergic sites in concert with other neurotransmitter systems: chemical sensitivity of fetal brain structures The caudorostral gradient of appearance of muscarinic cholinergic receptor sites is also encountered with nicotinic choline& sites which become detectable in lower brainstem and spinal cord from GD 12.*’ A similar caudorostral development has been noted for benzodiazepine binding sites3’ and for the GABAergic system.“* Studies on the ontogeny of ChAT-positive cells in forebrain areas of rats also show a slight caudorostral gradient of cholinergic neurogenesis.‘s*23 Since the outgrowth of cholinergic fibers is not known in detail, the relation between the development of these projections and the ontogeny of muscarinic binding sites remains uncertain. In cerebral cortex, we observed some overlap of ChAT-positive fibers penetrating the cortex with the localization of nicotinic cholinergic receptors.23 Different ontogenetic patterns are seen, e.g. with or neurotensin25 receptors. Combeta-adrenergic,” mon to all these developmental patterns is the fact that the distribution of receptor sites in the developing brain differs greatly from the adult condition and changes rapidly in relation to the developmental stage. AS a consequence, the main target areas of a drug also differ from the adult brain and change with ongoing development. In early fetal life, the lower brainstem, where a number of receptors are seen first, can be expected to be particularly sensitive
Fig. 8. Development of muscarinic choline@ binding sites in the fetal rat striatum and frontal neocortex at GD 18 (A, x 15.6), at GD 20 (B, x 14.3), and at GD 22 (C, x 13.7). frontal sections. In caudateputamen (cp) there is a lateromedial gradient of muscarinic choline& densities at all ages. Note also the appearance of a dotted immature distribution of binding sites at GD 22 in this brain region. At GD 18 and GD 20, some muscarinic sites are present in the neuroepithelium of the basal telencephalon (bn). In neocortex (nx), a clear ventrolateral to dorsomedial gradient of binding site densities is evident. s, Septum; sb, skull bone.
M.
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to chemical influences. An example is given by certain catecholamine neuron groups which possess cholinergic receptor sites early in development23 and are responsive to nicotine during fetal life.”
Acknowledgements--We
wish to acknowledge
Itic sklllul
technical assistance of Marline Saxer and the srcretarial help of Birgit Hofer. The study was supported by rhe Swiss National Science Foundation (grant no. 3.04%0.8’)
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