THE JOURNAL OF COMPAFWTIYE NEUROLOGY 305177-188 (1991)

Ontogeny of Somatostatin Receptors in the Rat Somatosensory Cortex BRUNO J. GONZALEZ, PHILIPPE LEROUX, CORINNE BODENANT, AND HUBERT VAUDRY Groupe de Recherche en Endocrinologie Moleculaire, CNRS URA 650, Unit6 AfFiliee a I'INSERM, Universite de Rouen, 76134, Mont-Saint-Aignan, France

ABSTRACT The distribution and density of SRIF receptors (SRIF-R) were studied during development in the rat somatosensory cortex by in vitro autoradiography with monoiodinated [TyroDTrp'IS14. In 16-day-old fetuses (E161, intense labeling was evident in the intermediate zone of the cortex while low concentrations of SRIF-R were detected in the marginal and ventricular zones. The highest density of SRIF-R was measured in the intermediate zone at E18. At this stage, labeling was also intense in the internal part of the developing cortical plate; in contrast, the concentration of binding sites associated with the marginal and ventricular zones remained relatively low. Profound modifications in the distribution of SRIF-R appeared at birth. In particular, a transient reduction of receptor density occurred in the cortical plate. During the first postnatal week, the density of receptors measured in the intermediate zone decreased gradually; conversely, high levels of SRIF-R were observed in the developing cortical layers (11 to VI). At postpartum day 13 (P131, a stage which just precedes completion of cell migration in the parietal cortex, the most intensely labeled regions were layers V-VI and future layers 11-111. From P13 to adulthood, the concentrations of SRIF-R decreased in all cortical layers (I to VI) and the pattern of distribution of receptors at P21 was similar to that observed in the adults. Detailed analysis of the results revealed i) a close association between the distribution of SRIF-R and the areas containing migrating neuroblasts and ii) a temporal correlation between the presence of SRIF-R and the transient expression of the neuropeptide SRIF in the developing somatosensory cortex. Taken together, our data suggest the existence of two populations of SRIF-R. a transient population, which could be implicated in the development of the parietal neocortex from E l 6 to P13, and a population of SRIF-R only expressed after the first postnatal week, which is likely to be involved in the process of neurotransmission. Key words: cortex development, receptor mapping, cortical plate, cortical subplate

Somatostatin (SRIF) is a tetradecapeptide widely distributed throughout the central nervous system (CNS) in both hypothalamic and extrahypothalalmic areas (Arimura et al., '75; King and Millard, '79; Bennett-Clarke et al., '80; Johansson et al., '84; Demeulemeester et al., '85; Beal et al., '87). In the rat, all the areas of the cerebral cortex contain SRIF-like immunoreactivity (SLI) (Bennett-Clarke et al., '80; Finley et al., '81; Johansson et al., '84; Vincent et al., '85). SRIF-containingperikaria are located in layers I1 to VI and to a lesser extent in the subcortical white matter. In the gray matter layers, SLI positive cells are usually less abundant in layer IV (Morrison et al., '83; Demeulemeester et al., '85; Laemle and Feldman, '85; Vincent et al., '85). SLI is associated with a variety of interneurons that can be

o 1991 WILEY-LISS, INC.

classified in two categories: large multipolar and medium to small bipolar interneurons (Morrison et al., '83; Laemle and Feldman, '85). In addition, a very abundant population of small bipolar weakly immunoreactive neurons has been observed (Meinecke and Peters, '86; Mizukawa et al., '87). Colocalization studies revealed that these cortical SRIFcontaining cells belong to a subclass of GABA-ergic interneurons (Somogvi et al., '84). Finally, a small proportion of SLI cells in the neocortex are pyramidal and modified pyramidal Accepted November 5,1990. Address reprint requests to Dr. Philippe Leroux, Groupe de Recherche en Endocrinologie Moleculaire, CNRS URA 650, Unite Afiiliee I'INSERM, Facult6 des Sciences, BP 118,76134 Mont-Saint-Aignancedex, France.

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binding sites on coronal sections of brains Fig. 1. Autoradiographic distribution of L1251-Tyro-DTrpRlS14 from 16- and 18-day-old fetuses. A. Autoradiograms depicting the distribution of somatostatin binding sites. B: Cresyl violet staining of the slices used to generate the autoradiograms. Scale bar = 1 mm. MZ, marginal zone; IZ, intermediate zone, VZ, ventricular zone; Cp, cortical plate.

cells (Morrison et al., '83; Laemle and Feldmann, '85). In particular, SLI has been detected in inverted pyramidal cells and Cajal-Retzius like neurons in layer I and deep layer VI (Ramon y Cajal-Agiierras et al., '89; Yamashita et al., '89). These cell types are particularly abundant during development (Luskin and Shatz, '85; Valverde et al., '891, and it is believed that they are involved in the maturation of the cortex (Chun et al., '87). The ontogeny of SRIF in the cortex has been investigated using both immunocytochemistry and hybridization techniques (Shiosaka et al., '81; Hayashi and Oshima, '86; Cavanagh and Parnavelas, '88; Naus et al., '88). These studies revealed that SRIF is expressed early during development. SRIF inRNAs have been visualized as early as embryonic day 16 (E16) (Naus et al., '88), while SRIFcontaining cells have been detected in the visual cortex at E l 7 (Eadie et al.,'87). In rat or cat fetuses, SLI-containing cells are located in the intermediate zone of the cortex, especially in the subplate layer, and to a certain extent in the marginal zone (Chun et al., '87; Eadie et al., '87; Naus et al., '88; Chun and Shatz, '89). These neurons, which belong to the earliest generated neurons (between E l l and E l 3 in the rat) (Readler and Readler, '78) and which present characteristics of differentiated cells such as dendritic

arborisation and synaptic contacts (Briickner et al., '76; Chun and Shatz, '891, disappear when corticogenesis is completed (Shatz et al., '88). It has been proposed that this transient cell population is involved in the establishment of corticofugal connections that serves to guide the future corticopetal innervation (McConnell et al., '89). The existence of the subplate zone also favors interactions between migrating neurons and populations of ingrowing affererits (Rakic, '88). While in the adult cortex all regions, including layers V and VI, are innervated by a dense SRIF-containing network (Naus et al., '88; DeLima and Morrison, '891, in fetuses and newborn rats, SLI fibers are confined to t.he TABLE 1. Concentrations of ['"I-Tyra-DTrp81S14 Binding Sites in Various Layers of the Parietal Cortex From 16- and 18-Dav-OldFetuses Bmax (fmol./mg prot.) Cortical layers

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intermediate zone or corpus callosum (0-O), and the ventricular zone (A-A). Each count was made on 8 pm wide field. B: Density of silver grains counted in the intermediate zone or corpus callosum (0-0) and the developing cortical layers I1 to VI (.---I).

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bindFig. 3. Autoradiographic distribution of ['Z51-Tyrn-DTrpa]S14 ing sites on coronal sections of the parietal cortex from 16-day-old fetuses. A: Photomicrograph of the cresyl violet stained slice used to generate the autohistoradiogram. B: Darkfield micrograph showing the distribution of silver grains (total binding). C : Concentrations of silver grains quantified in the various layers of the developing parietal cortex.

The values represent specific binding, i.e., total number of grains per field minus number of nonspecific grains counted on a consecutive section, which had been incubated with an excess of unlabeled somatostatin. MZ, marginal zone; IZ, intermediate zone; VZ, ventricular zone. Scale bar = 0.25 mm.

intermediate and the marginal zones (Naus et al., '88). In particular, the cortical plate (which gives rise to layers I1 to VI) exhibits very few SLI nerve terminals during the perinatal period. The distribution of SRIF receptors (SRIF-R) has been studied in the mature CNS of various species (Epelbaum et al., '85; Leroux et al., '85; Reubi and Maurer, '85; including man (Uhl et al., '85). In the neocortex, the highest density of SRIF-R is located in the deeper layers V and VI, while layers I to IV are weakly labeled (Leroux et al., '85). In a recent study, we have shown that the brain of fetuses and young rats contains high concentrations of SRIF-R (Gonzalez et al., '89). In the cerebellum, these receptors are expressed in the external granule cell layer, a transient proliferative and premigratory area (Gonzalez et al., '88). While the developmental profile of SRIF biosynthesis and distribution in the rat cortex has been well studied, nothing is known regarding the ontogeny of SRIF-R. Obviously, a clear understanding of the functional role of SRIF during the development of the cortex requires that the location of both SRIF-containing neurons and SRIF-R is determined.

In the present study, we investigated the distribution of SRIF-R in the developing parietal cortex. In order to provide precise information on the density and location of SRIF-R in the somatosensory cortex, the density of receptors has been measured by direct visual grain counting in emulsion coated slices.

METHODS Animals Adult Wistar rats were maintained at 22 t 1°Con a daily 12-hour light, 12-hour dark cycle, with free access to f%od and water. The morning when sperm was found in the vaginal smear was considered day 1of pregnancy. Rats were killed on days ranging from fetal day 16 (E16) to day 21 postpartum (P21). Pregnant females were killed by decapitation without prior anaesthesia. Fetuses, newborns (NB), and pups (P4 to P8) were decapitated and the whole heads were snap-frozenin isopentane at -30°C. From P8 onward, brains were dissected out and frozen in isopentane. All brains were kept at -80°C until use for autoradiography.

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binding sites on coronal sections of the Fig. 4. Autoradiographic distribution of ['251-Tyro-DTrps]S14 parietal cortex from 18-day-old fetuses. CP, cortical plate. For details, see legend to Figure 3.

Chemicals Synthetic somatostatin 14 6 1 4 ) was provided by Dr. J. Chanteclerc (SANOFI, Paris, France). [Tyro-DTrp8]S14was a gift of Dr. D. H. Coy (New Orleans, LA). The ligand was radiolabeled by means of the lactoperoxidase technique as previously described (Laquerriere et al., '89) and purified by reverse phase HPLC on a Zorbax C18 column (25 x 0.4 cm, Merck, Darmstadt, FRG) using a gradient of acetonitrile in triethylammonium phosphate buffer (0.25 M, pH 3 ) . Monoiodinated [1251-Tyro-DTrps]S14 eluted at 26% acetonitrile. The specific radioactivity of the tracer was approximatively 2,000 CiimMole.

Slice binding studies Unfixed heads or brains were sliced on a cryostat (Frigocut, Reichert-Jung, FRG) at 20 pm, in frontal planes. Sections were thaw-mounted on gelatin-coated slides, dehydrated under vacuum, and kept frozen at -80°C for slice binding, as previously described (Leroux et al., '85). Briefly, slices were incubated with 32 pM ['251-Tyra-DTrp8]S14, and nonspecific binding was determined in adjacent sections incubated with the radioligand in the presence of 1 p.M unlabeled S14. After a 60 minutes incubation, all slices

were washed three times ( 5 minutes each) in ice-cold buffer and dried under a stream of cold air.

Film autoradiographic studies Radioactive slices were apposed to ['HI Ultrofilm (LKB) for 3 weeks. After exposure slices were stained by cresyl violet. Autoradiograms were quantified by means of a computer-assisted image analysis system BIO 500 (BIOCOM, Les Ulis, France).

Emulsion-coated autoradiographic studies Radioactive slices were dipped into Kodak NTB-2 liquid emulsion at 40°C. After a 4-week exposure, the autohistoradiographic preparations were counter-stained with toluidine blue and quantified by direct visual grain counting under light microscope (Leitz, Orthoplan) at large magnification ( x 1250 under oil immersion). Three or four animals were used for quantification at each stage of development.

RESULTS In the present study, the nomenclature of the Boulder Committee ('70) has been used for the various histological layers of the developing neocortex.

B.J. GONZALEZ ET AL.

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Fig. 5. Autoradiographic distribution of ['2sII-Tyro-DTrpRlS14 binding sites on coronal sections of the parietal cortex from newborn rats. For details, see legend to Figure 4.

At E16, the highest density of silver grains was observed in the intermediate zone, while the lowest concentration Typical autoradiograms obtained at E l 6 and E l 8 are was measured in the ventricular zone near the border of the shown in Figure 1. The concentrations of [1251-Tyr0, lateral ventricle (Fig. 3). In this area, the number of grains DTrps]S14 binding sites, measured in the different layers of gradually increased towards the intermediate zone (Fig. 3). the immature neocortex, are indicated in Table 1. High Conversely, the concentration of silver grains observed in levels of SRIF-R were found as early as E l 6 in the the intermediate zone decreased in the vicinity of the intermediate zone, while low densities were observed in the marginal and the ventricular zones. At E18, the intermedi- marginal zone (Fig. 3). At E18, a robust increase of the labeling intensity was ate zone still exhibited the highest concentration of binding sites; at this stage of development, moderate levels of observed in the developing neocortex (Fig. 2A). The augmenreceptors occurred in the cortical plate. However, the tation was mainly associated with the intermediate zone, resolution of the autoradiographic films was too poor to which contained the highest density of grains (Fig. 2B and discriminate amongst the binding observed in the various 4). The concentration of grains slightly increased in the layers of the developing neocortex with sufficient accuracy. ventricular zone and remained very low in the marginal zone (Fig. 4).In the cortical plate, which first appears at Emulsion autoradiography E18, a dense labeling was noticed in the inner part. Marked changes appeared from E l 8 to birth (Figs. 4 and In order to validate the method of quantification on emulsion-coated autoradiography, we have first examined 5). In particular, the density of silver grains in the intermethe correlation between the density of silver grains quanti- diate zone and the cortical plate decreased by 56% and 37%, fied by direct visual counting, and the concentration of respectively (Fig. 2B). However, as the thickness of the SRIF-R measured by the image analysis system. A linear cortical plate undergoes a 4-fold increase during the last 3 relation was obtained with a coefficient of correlation of days of fetal life, the number of grains in the cortical plate 0.93 indicating that both methods provide identical results. actually increased during this period (Fig. 2A). In fact, the The number of silver grains was measured at different prenatal period appears as a transitional stage. In fetuses, stages (from E l 6 to adulthood) in the various cortical zones the highest density of labeling was seen in the intermediate zone while, after birth, the concentration of silver grains (Fig. 2A).

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B.J. GONZALEZ ET AIL.

184 was higher in the developing cortical plate (Fig. 2B). From E 18 to birth, no significant modifications were observed in the ventricular and the marginal zones that still contained very few silver grains. At P4, the number of grains observed in the somatosensory neocortex markedly increased (Fig. 6). However, this augmentation concerned only the developing cortical plate, as 70% of the silver grains were located in this region (Figs. 2A and 6). The highest density of grains was measured at the interface between layer VI and the rest of the developing cortical plate (Fig. 6). In the intermediate zone, the fall of silver grain density (as illustrated in Fig. 2B), was heterogeneous. Grains remained more numerous in the inner part of the layer (Fig. 6). In the marginal zone (future layer I), the concentration of silver grains slightly increased from birth to P4 (Fig. 6). At this stage, the thickness of the ventricular zone was markedly reduced. At P8, the number of silver grains continued to increase and 89% of them were detected in the developing cortical layers (I1 to VI; Fig. 2A). The concentration of silver grains appeared homogeneous in layers I to VI, except in the differenciating layer IV that contained a lower density (Fig. 7). In the intermediate zone (future corpus callosum), the decrease of grain density initiated at P4 was intensified in the whole layer (Fig. 2B and 7). At this stage, the ventricular zone has almost completely disappeared.

The number of silver grains peaked at P13 (Fig. 2A.). Slight changes were observed in the distribution of labeling between P8 and P13. In particular, the silver grain density decreased in the inner part of layer VI and increased in the outer margin of the same layer (Fig. 8). In addition, the fall of silver grain concentration initiated at P8 in layer IV was more obvious at P13. Finally, the density of grains continued to decrease in the corpus callosum (Fig. 8). From P13 to adulthood, the absolute number of silver grains counted in the somatosensory cortex markedly decreased in all cortical layers (Fig. 2A). At P21, very few grains were seen in the corpus callosum (Fig. 9). Between P21 and adulthood, only minor modifications were observed, i.e., a slight decrease of the grain density in layers 11, 111, and IV (Fig. 2B, 9, and 10).

DISCUSSION The present study describes the distribution of SRIF binding sites in the rat somatosensory cortex during preand post-natal life. Previous studies support the not,ion that SRIF binding sites expressed in the immature rat brain correspond to authentic receptors (Chneiweiss et al., '85; Gonzalez et al., '89). Binding experiments using brain membrane preparations indicate that these receptor sites are already regulated by GTP in 15-day-old fetuses (Gonzalez et al., '89). Furthermore, SRIF appears to be a

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Fig. 8. Autoradiographic distribution of ["sII-Tyro-DTrp*1S14 binding sites on coronal sections of the parietal cortex from 13-day-old rats. For other details, see legend to Figure 7.

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Fig. 9. Autoradiographic distribution of ['251-Tyra-DTrp81S14 binding sites on coronal sections of the parietal cortex from 21-day-old rats. For other details, see legend to Figure 7.

potent inhibitor of adenylate cyclase activity in embryonic cortical cells in primary culture (Chneiweiss et al., '85). Taken together, these results indicate that SRIF-R are coupled to G-proteins and potentially functional during fetal life. When compared with previous data on the distribution of SRIF in the developing rat neocortex, our results reveal a good correlation between the temporal expression of SRIF-R and the occurrence of the neuropeptide (McDonald et al., '82; Eadie et al., '87;Naus et al., '88). During the histogenesis of the somatosensory cortex, SRIF-R are first detected at E l 6 and the highest number of binding sites is found at P13. Similarly, the mRNAs encoding for SRIF are detected as early as E l 6 and the expression rate increases until P15 (Naus et al., '88). During fetal life, SRIF-R are mainly located in the intermediate zone while moderate to low densities are visualized in the marginal and ventricular zones and in the cortical plate. At E20, a large number of SRIF-immunoreactive cells is observed in the intermediate zone (Naus et al., '88),particularly in the upper part termed the subplate (Kostovic and Molliver, '741, while fibers are abundant in the intermediate and marginal zones (Naus et al., '88). During the first postnatal week, the density of SRIF-R measured in the intermediate zone is markedly reduced. In contrast, high concentrations of SRIF-R are found in the developing cortical plate. In the same period, the number of S28(1-12)-containing cells and the expression of SRIF mRNAs per neuron increase in the subplate

(Naus et al., '88). In addition, at the end of the first postnatal week, a new population of S28 immunoreactive cells appears in layers VI and V (Naus et al., '88).At P13, a dramatic fall of SRIF-R is seen in all cortical layers and the pattern of distribution of receptors at P21 is similar to that observed in the adults. The regression of SRIF-R in the prepubertal period is correlated with the dramatic decrease of S28(1-12) immunoreactivity observed at P12 (Naus et al., '88). New fibers immunoreactive for S28(1-12) reappear after P30, particularly in layers VI and V; these fibers probably originate from the elongation of processes emerging from SRIF-containing GABA-ergic interneurons (Naus et al., '88). The first population of SRIF neurons observed by Naus et al. ('88) from E20 to P12 may belong to the population of early differentiated peptidergic neurons described in the developing neocortex of the cat (Chun et al., '87; Chun and Shatz, '891, which were identified as CajalRetzius neurons (Ramon y Cajal-Agiieras et al. '89). In the cat neocortex, most of the peptidergic neurons that express SRIF are present in the subplate. However, as the brain matures, these cells disappear when migrating neurons and subcortical axonal processes invade the cortical plate (Chun The important decrease of S28(1-12) immunoet al., '87). reactive neurons and fibers, observed at P12, occurs during the process of cell death in the neocortex, when cell migration is completed (Luskin and Shatz, '85; Rakic, '88). The data presented herein show that a first population of SRIF-R is expressed from E l 6 to P13 coincident with the

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binding sites on coronal sections of the Fig. 10. Autoradiographic distribution of ['2sII-Tyro-DTrp81S14 parietal cortex from adult rats. For other details, see legend to Figure 7.

appearance of a transient SRIF-ergic innervation. They also indicate that. the intermediate zone contains the first target cells for SRIF. These observations support the notion that SRIF may control cell migration and/or regulate the establishment of cortical projections by the transient cells of the subplate (Kostovic and Rakic, '80; McConnell et al. '89). Detailed analysis of the autoradiograms revealed a close association between the distribution of SRIF-R and the areas containing migrating neuroblasts. This concept is schematically presented in Figure 11. During fetal life, SRIF-R are mainly located in the intermediate zone (Fig. 11B). In the same way, the first migrating neurons of the future layers VI and V, which are generated between E l 2 and E16, are located in the intermediate zone from E l 6 to E20 (Berry and Rogers, '65) (Fig. 11A). These cells reach the future layer VI between E20 and birth. At birth, SRIF-R are distributed both in the intermediate zone and the cortical plate. While at birth, migrating neurons of the future layers VI and V have already reached the cortical plate, the cells of the future layers IV, 111, and 11, generated between E l 7 and E20, are still located in the intermediate zone (Berry and Rogers, '65; Briickner et al., '76) (Fig. 1lA). From P4 to P8, the density of receptors is markedly reduced in the intermediate zone, and the highest concentration of SRIF-R is located in the developing cortical layers (Fig. 11B).At this period, the latest generated neurons have left the intermediate zone and undergo their migration

through the developing cortical layers VI and V (Berry arid Rogers, '65) (Fig. 11A) while transient neurons are still present in the intermediate zone (Naus et al., '88). At P13, the distribution of SRIF-R is quite similar to that observed in adult and the cell migration is almost completed. After P13, the abrupt fall of SRIF-R observed in the neocortex coincides with the end of cell migration. These data suggest that SRIF-R expressed during the histogenesis of the neocortex belong to a transient population of receptor sites that are worn by migrating neurons. In conclusion, our study reveals that two populations of SRIF-R appear sequentially in the rat cerebral cortex. These observations are in good agreement with the previous reports that suggest the existence of two different populations of SRIF-containingcells during the ontogeny of the somatosensory cortex (Feldman, '88; Naus et al., '88). The first population is composed of transient neurons located in the intermediate zone, which may activate SRIF-R expressed between E l 6 and P13, in particular in those cells undergoing migration. These receptors vanish at the end of histogenesis of the neocortex (P13) in coincidence with the disappearance of the S28(1-12) immunoreactive cells and fibers. The second population of SRIF-producing cells is present from P7 onwards in layers VI and V (Naus et al., '88).These neurons probably correspond to the subpopulation of GABA-ergic interneurons immunoreactive for SRIF found in the adult cortex (Somogyi et al., '84). Taken

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Fig. 11. A: Schematic representation of the migratory pattern of neuroblasts during the period of histogenesis in the rat cerebral cortex (modified from Berry and Rogers, '65). 0,7 , 0,0 , represent the relative positions of cells after labeling on the 16th, 17th, 18th, and 19th to 20th days of gestation, respectively. B: Schematic representation of the silver grains during the development of the somatosensory

cortex. Clear and dark greys represent high and moderate concentrations of silver grains, respectively. Black represents non specific level of silver grain concentrations. MZ, marginal zone; CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone; CC, corpus callosum, I to VI, cortical layers I to VI.

together, our data support the concept that SRIF may act as a neurotrophic peptide that exerts pleiotropic activities during histogenesis of the CNS. Depending on the brain structures. SRIF might be involved in different developmental processes including i) cell migration in the somatosenneocortex as suggested in the present study, ii) cell division as illustrated in the external granule cell layer of

the cerebellum (Gonzalez et al., '88), and iii) maturation of corticospinal pathways (Charnay et al., '88).

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ACKNOWLEDGEMENTS This work was supported by the Ministere de la Recherche et de la Technologie (grant number 87.M.0074) and the

B.J. GONZALEZ E T AL.

188 Conseil Regional de Haute Normandie. B.J.G. and C.B. were recipients of fellowships from the Ministere de la Recherche et de la Technologie.

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