Developmental patterns of somatostatin-receptors and somatostatin-immunoreactivity during early...

Neuroscience Vol. 62, No. I, pp. 317-325, 1994 Elaevier Science Ltd

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DEVELOPMENTAL PATTERNS OF SOMATOSTATIN-RECEPTORS AND

SOMATOSTATIN-IMMUNOREACTIVITY DURING EARLY NEUROGENESIS IN THE RAT

E. MALJBERT,* A. SLAMA,f P. CIOFI,§ C. VIOLLET,$ G. TRAMU,Il J. P. DUPOUY* and J. EPELBAUM$t

*Laboratoire de Neuroendocrinologie du DBveloppement, Universite de Lille I. 59655 Villeneuve d’Ascq Cedex, France

SINSERM. Unitt 159, 2 Ter rue d’Alesia, 75014 Paris Cedex France IlLaboratoire de Neurocytochimie Fonctionnelle, CNRS. URA 339, Universite de Bordeaux 1. 33405

Talence Cedex, France §INSERM. Unite 156, Place de Verdun, 59045 Lille Cedex, France

Abstract-The temporal pattern of distribution of somatostatin receptor was investigated using the somatostatin analogue [‘2SI]Ty~-DTrps-somatostatin,, as a ligand and compared with that of somatostatin immunoreactivity during early developmental stages in the spinal cord and the sensory derivatives in rat fetuses. Qualitative and quantitative analysis showed that somatostatin receptors were detected in a transient manner. In the neural tube, they were clearly associated with immature premigratory cells and with the developing white matter. During the time-period examined (from day 10.5 to 16.5), the disappearance of somatostatin receptors followed a ventro to dorsal gradient probably linked to the regression of the ventricular zone. In sensory derivatives, they were expressed in the forming ganglia and their central and peripheral nerves from embryonic day 12.5 to 16.5 inclusive, with a peak around day 14.5 and low levels observed at day 16.5. Competition experiments performed at embryonic day 14.5 demonstrated that somatostatin,_,,, somatostatin,,,. and Octreotide displaced specific binding with nanomolar affinities while CGP 23996 was only active at micromalar doses. Such displacements are compatible with the SSTR2 and/or SSTR4 pharmacology. During the time period examined, some transient somatostatin immunoreactive cell bodies and fibers were detected in the neural tube and in the sensory derivatives.

These results demonstrate the existence. in neuronal derivatives, of a complex temporal and anatomical pattern of expression of somatostatin receptors, from the SSTRZ/SSTR4 subtype(s), and somatostatin immunoreactivity. It appears that the transient expression of somatostatin receptors and/or somatostatin immunoreactivity characterizes critical episodes in the development of a cohort of neurons; a fact that unequivocally reinforces the notion that somatostatin plays a fundamental role during neurogenesis in vertebrates.

A transient expression of somatostatin-immunoreactivity (IR) is a characteristic feature of developing neurons during periods of neurogenesis and differentiation of the brain~6~9~“~‘3.32~38~-2 the spinal cord,2’ the sensory derivatives22x’9 and the sympathetic ganglia. ** In the rat optic pathway&’ and cerebellum”‘8~44 as well as in the human spinal cord,’ a transient expression of somatostatin-receptors was also reported during neurogenesis. These observations have led to the assumption that somatostatin may play trophic or synaptogenetic roles during some

developmental processes.

In the rat, it is established that between embryonic

tTo whom correspondence should be addressed. Abbreoiations: CGP 23996, Ciba Geigy Peptide 23996;

DRG, dorsal root ganglia, E, embryonic day, GAD, glutamate decarboxylase; IR, immunoreactivity; VZ, ventricular zone.

days E10.5 and 16.5 critical events determine the differentiation of neurons in the spinal cord’,3,*,‘4’~36.43 and the sensory derivatives.24.29 As demonstrated by Ho, *’ Katz et CI~.*~ and u&29 somdto- statin-IR is transiently expressed during this period in spinal cord neurons, cranial ganglia and dorsal root neurons. However, the temporal pattern of somatostatin-receptors during these early developmental stages remains to be established. The aim of the present study was to determine, using the iodinated non-degradable somatostatin analogue [‘251]Tyro- DTrp*-somatostatin,,, whether the spinal cord and the sensory neurons express somatostatin-receptors between embryonic days 10.>16.5. Since cDNA for five different somatostatin receptor subtypes have recently been characterized (for review, see Ref. 10) a preliminary pharmacological characterization was also undertaken.

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318 E. Maubert er ui.

EXPERIMENTAL PROCEDURES

Animals

Adult female Wistar albino rats (Iffa~redo, L’Arbesle, France) were maintains under standard conditions of temperature (23 & 2°C) and lighting (12j12 h light/dark schedule) with water and food available ad iibitum. Females were exposed to males for one night. If spermatozoa were found in the vaginal smears in early morning the following day, it was considered to be EO. Entire embryos were collected at 12.00 h from a& least three pregnant females killed daily from El05 to E16.5. Groups of five to 10 developing embryos per stage were studied. The troncal regions from older stages were also investigated in some fewer number of older embryos, pups and adults.

Receptor labeling was performed as previously de- scribed3’ with minor modifications. Briefly, specimens were frozen on dry-ice and cut on a cryostat. Sixteen-micrometer- thick sections were collected on gelatin-coated slides and stored at -80°C until use. Slides were then brought to room temperature and preincubated for 15 min in 50mM Tris- HCl buffer pH 7.6, containing 0.25 mM sucrose and 0.2% bovine serum albumin. The slides were then incubated for 45 min at room temperature in the above buffer supplemented with 0.1-0.5 nM [‘Z5i]Tyro-DTrp*-somatostatin,,, bacitracin (20 mg/l) and MgCl, (I g/l), To determine non- specific binding, adjacent sections were incubated in the same solution supplemented with 1 pM of somatostatin,,. Competition experiments were performed on triplicate thoracic sections from different E14.5 fetuses using somatostatin,,, somatostatin,,, Octreotide and CGP 23996 in in- creasine concentration from IO-” to 10e6M. Slides were then wished twice in cold buffer (2 x 5 min) and ligands were irreversibly linked to their receptors by covalent bind- ine induced bv a 30min fixation in a cold solution of phosphate buher (50mM) pH 7.6, containing 4% glu- taraldehyde. Sections were then dried and apposed onto Hyperfilm b-max (Amersham) for three days at 4°C. After this period, films were removed, revealed with Kodak Dektol developer and fixed in Kodak 3000 fixer. The sections were dehydrated and dipped into Ilford, K5 liquid emulsion at 40°C and exposed for 15-21 days at room

temperature. Emulsions were then revealed as for films, and sections were counterstained with Cresyi Violet. Binding was yuantitated by reference to iodinated standards pre- pared from brain paste with the help of a computer assisted image analyser using a camera and the RAG program (Biocom, les Ulis, France) which allows for conversion of optical densities into radioactivity units’s Com~tition curves were analysed by non linear regression using the Parker and Waud model)” which allows for statistical evaluation of dissociation constant kinetics.

lmmunokistofluorescenre technique

lnlmunohisto~uores~nce for somatostatin was performed in parallel to receptor binding but on different

fetuses from the same mothers at the respective gestational stages. Specimens were fixed overnight by immersion in a 0.2?‘0 picric acid-2% paraformaldehyde fixative and then rinsed in a Verona1 buffer containing 20% sucrose. as previously described. 29 Whole embryos were cut transver- sally and sagittally on a cryostat. Sections 5 ~IO~m m thickness were processed for immunofluorescence as previously described29 using a rabbit antiserum to somatostatin 28,_,2 (coded as AS7).

In some instances, howeever, immunohistochemistr~ was performed on sets of cryostat sections of fresh frozen material collected for radioautographic studies (El 3.5 and E15.5). In this case, sections mounted on slides were fixed by a 4-h immersion in paraformaldehyde fixative and processed for immunohistochemistry as described above following copious rinsing in Verona1 buffer.

RESULTS

General comments

Histologic preservation and immunohistochemical staining on the fresh-frozen, immersion-fixed sections were of considerably lesser quality than those obtained in regularly fixed embryos.

In radioautographic studies a similar distribution of somatostatin-receptors was observed on films and liquid emulsion dipped shdes (cf. Figs 1B and 2A). Sensitivity was greater in the latter case. Namely, specific somatostatin-receptors in the cephalic neural tube and in the dorsal root ganglia (DRG) were first detected at El 1.5 and E12.5, respectively, on dipped slides, whereas no signal could be observed in these structures on films previously exposed to the same sections.

The development and organization of the ectoder- mic and neural crest derivatives follow a rostra1 to caudal temporal sequence, i.e. embryonic rostra1 regions organize earlier than caudal ones. Such an

anatomical and temporal sequence of events was observed for the expression of somatostatin receptors and IR. Although we have studied the cephalic-to- lumbar portion of the embryos, most of our descrip- tions and illustrations focus on events concerning the troncal portions, in order to obliterate the natural asynchrony in development of regions distant from each other along the rostrocaudal axis.

Neural tube

In the neural tube. somatostatin receptors were not detected before El 1.5. At this stage, they appeared to

Fig. 1. Distribution of [‘251]Tyro-DTrp*-somatostatin,,-receptors in the developing neural tube, as apparent from film radioautograms directly projected onto photographic paper. (A-E) Frontal sections through the thoracic neural tube at embryonic days 12.5 (12.5; A,, total and A,, non-specific binding), E13.5 (B), E14.3 (C), El55 (D) and E16.5 (E). Neural tube: at E12.5-13.5 somatostatin receptors are present in the lateral but not medial (asterisks in A, and B) regions of the neural tube. From E12.5 to El45 intense labeling is present over the basal (arrows in A, and B) and intermediate (arrows in C) plates. Labeling is also observed over the white matter, especially over the ventral funiculus (arrowheads in B-D). Homoge- neously distributed over the neural tube at early stages (A, and B), the labeling is deeper in the dorsal vs ventral regions at later stages (C-E). Dorsal root ganglia: Somatostatin receptors are detected in the dorsal root ganglia (double-headed arrows) and their central or peripheral processes (thin double-arrows) from E13.5 to 16.5 (B--E). Labeling is strongest at E14.5. Feathered-arrows in C and E point to labeling

over the lungs primordia. A x 38, B x 48; C x 44; D x 33; E x 22.

_ . . c somatostatin receptors 319

Fig. 1.

Fig. 2, Comparison of the distribution of ~‘L’I]Tyro-DTrp”-somatostatj~~,~- receptors (A and D, brightfields) and somatostatin-immuno~uo~scence (B, C) on fronlsl cryostat sections through the thoracic neural tube from diKerent fetuses at identical developmental stages (A and B, E13.5: C and D, E15.5). Identical symbols in A and 9, or C and D, point to same structures. (A,B) The mediodorsal parts of the epcndqmal zone (asterisks) is devoid of both somatostatin receptors and somatostatin-jmmunor~ctivity; the ventral horns (thin double-arrows) display low binding levels and contain somatostatin-IR cells and processes: the oval bundle of His (double-headed arrows) and the DRG (straight arrows) present both signals; the basal plate (arrowheads) is strongly radiolabeled and ~matostatin-immunone~tive. The feathered arrow in B points to a peripheral nerve containing somatostatin-iR axons. (C,D) At this low magmfication, somatostatin-IR appears to be essentially present in nerve fibers in the dorsal horns (double-headed arrows) and in presumed commissural neurons and their corresponding processes (arrows). Both somatostatin-R and somatostatin-IR elements are distributed in and around the ventral funicu!us (arro\rheads). Neurons in rhe ventral horns are not somatostatin-IR (asterisk) at this stage. Radioauto- graphic signal is stronger in dorsal than ventral regions of the neural tube and is also present m the dorsal

root ganglia (star). A Y 153: B x 135: C x 190: D x 125,

320

Transient expression of samatostatin receptors 321

Fig. 3. Fluorescence micrographs of frontal cryostat section of E13.5 rat embryo showing the expression of somatostatin-immuRore~tivity in the neural tube. Somatostatin-IR within neuronal cell bodies and fibers in the ventral horn (short thick arrows). The arrowhead in the lower right side of the figure points to the spinal nerve which seems to receive somatostatin-IR fibers from both ventral horn neurons

(double-headed arrow) and dorsal root ganglia (thin arrows). x 430.

be associated with the lateral regions of the cephalic neural tube. From El 1.5 on, somatostatin receptors were then detected across the borders of the ventricular zone (VZ), and more laterally in the developing white matter, throughout the dorsoventral and rostrocaudal extents of the neural tube. Dorsally, at early

stages (El2.5-13.5) the centralmost parts of the VZ did not display detectable levels of somatostatin receptors (Figs lA, B, 2A). In lateral parts of the dorsal neural tube, somatostatin receptors were detected from around El 1.5 and through E16.5, with an intensity increasing up to around El4.5-15.5 and decreas- ing thereafter (Fig. 1).

From E12.5 on, an intense labeling was observed over median bilateral areas in the ventral or intermediate neural tube. These strongly labeled areas appeared to correspond to clusters of immature cells occupying the basal plate region at El2.513.5, and the inter-

mediate plate region at El4.5-15.5 (Figs IA-C, 2A). Comparati~ei~, lower levels of somatostatin receptors were vizualized in the developing ventral horns from E12.5 on (Figs I9 2). In addition, between E12.5 and 16.5, somatostatin receptors were present in the developing white matter (Fig. 1).

Around birth the neural tube displayed a labeling of homogenous distribution and low level never match-

ing in intensity that observed at earlier stages (Fig. 4). Somatostatin-IR was clearly detected around El 1.5

in nerve fibers and cell bodies of the developing ventral horns. In contrast to the distribution of somatostatin receptors, extending to the VZ and even crossing its border, all these somatostatin-IR cell populations were always located lateral to the VZ. Somatostatin- IR was continuously detected over the time-period

investigated in various populations of cell bodies and fibers having a differential distribution with respect to the developmental stages and spinal levels. For instance, in the thoracic spinal cord, some presumptive motoneurons were seen to intensely express somatostatin-IR from El15 to E13.5 (Figs 2B and 3), while some putative commissural neurons were observed to be somatostatin-IR from El4.5-16.5 (Fig. 2C). In every instance there were somatostatin-IR varicose fibers seen in the developing gray matter, intermingled or not with sornatostatin-IR cell bodies. In addition, somatostatin-IR nerve fibers were observed streaming along the borders of the VZ, with few of them appear- ing to intervene between cells of the VZ. From El 2.5 to

14000

12000 --O- NTd

B 10000 -)- NTv

3 8000 --o-- DRG

2 6000

4000

2000

Fig. 4. Quantification of somatostatin receptors in neural tube and dorsal root ganglia during development. Each dot is composed of three to six animals except for the PlO/aduIt group which is composed of two ammats. Each value represent means + S.E.M. of somatostatin receptors levels express m disintegrations/min (d.p.m.)/surface units (d.p.m.!su). NTd, dorsal neural tube; NTv, ventral neural tube. *P < 0.5 vs respective El0 and postnatal day IO/adult.

at least E16.5. somatostatin-IR was observed in varicose fibers distributed throughout the developing white matter, as clearly apparent in the ventral funiculus from frontally sectioned embryos (Fig. 2C).

Sens0v.v derivatives

During sensory neurogenesis, somatostatin receptors were detected from rostra1 (trigeminal, jugular superior and nodose ganglia) to caudal (DRG) sensory derivatives. Both somatostatin-IR and somatostatin receplors were transiently expressed during embryonic development in the regions of the sensory ganglia and their central and peripheral projections (Figs IB-E, 2A,B).

As revealed by our qualitative or quantitative analysis (Fig. 4). the distribution and eventual overlap of both types of signal in the DRG were, from a chrono- logical viewpoint, more complex in these derivatives than in the neural tube. For example, somatostatin-IR was faintly expressed in DRG neurons at El 1.5 whereas somatostatin receptors could not be clearly visualized over these structures before E12.5. Both signals overlapped in distribution from E12.5-14.5, with an apparent maximal expression between El 3.5 and E14.5, for somatostatin-IR and somatostatin receptors, respectively. In cell bodies, both signals could not be detected after E16.5 but some scarce somatostatin-IR nerve fibers were still observed in the DRG at E16.5. The examination of immunostained sections from embryos at developmental stages E13.5 and E15.5 and adjacent to those sections used for radioau- tography allowed us to ascertain that somatostatin receptors and somatostatin-IR were indeed expressed in the same areas containing cell bodies and fibers (DRG at E13.5) or solely nerve fibers such as the oval bundle of His (E13.5 embryo) and the ventral funiculus (E15.5) (not shown).

PhurmacologicaI rharac‘terizution

As shown in Fig. 5, somatostatin,,, somatostat&, and Octreotide were approximately equipotent (in the nanomolar range) to displace [“‘I]Tyr’-DTrpX- somatostatm,, binding either in the dorsal part of neural tube or in the DRG from E14.5 embryo. In contrast, CGP 23996 was only active at micromolal concentrations.

UISCUSSION

General comments

The present work provides evidence for the transient occurrence of somatostatin receptors belonging to the SSTR2 and/or SSTR4 subtype(s) in the spinal cord and the sensory derivatives during early rat neurogenesis. Furthermore, our results suggest that as already postulated for that of somatostatin-IR,*’ the transient expression of somatostatin receptors may also be a common feature of various neuronal cell types. A wealth of experlmental data suggests the involvement

Neural lube -

80

60

Concentration (M)

Dorsal root ganglia

120

100

80

60

40

20

0

Somatostatin t-14

Somatostatin I-2X

Octrwtide

CGP 23996

Concentration (M)

Fig. 5. Deplacement of specific [“‘I]Tyr”-DTrpH-somatostatin,, by somatostatin,,, somatostatinz,, Octreotide and CGP 23996 in the dorsal part of the neural tube (upper panel) and in dorsal root ganglia (lower panel) at E14.5. Results are expressed as o/D of deplacement of specific binding. Values are means of nine measurements obtained from three frontal thoracic sections for each doses. IC,,s were similar in neural tube and spinal ganglia for each analogue (respectively. somatostatin,,: 0.78 5 0.05 and 0.79 + 0.48; somatostatin>,: 3.72 k 0.39 and 2.44 + 0.3; Oc- treotide: 2.07 * 0.74 and 3.8 + 1.79 and CGP

23996 z 300 nM).

of the peptide somatostatin in important developmental processes. A necessary step to ascribe a physiologi- cal role for somatostatin during development is to demonstrate the presence of the receptor(s) for the molecule together with the peptide itself in a given tissue at a given developmental stage. This has been achieved by various studies of the cerebellar cortex in which both somatostatin receptors and somatostatin- IR are transiently expressed during the early postnatal period (review in Ref. 18). In this study. we have extended these results by showing the distribution of both signals in neuronal derivatives at earlier developmental stages.

Before discussing any aspect of our findings, it is necessary to point out our inability to discriminate the individual cell types that expressed somatostatin receptors during the time period investigated. This is particularly relevant to the interpretation of the results of the study reported here since, during development of the nervous system, neuronal cells, glial cells and Schwann cells are concurrently generated, and because neuroneuronal as well as neuroglial interactions are known to occur during the organization of the nervous tissue (review in Ref. 19). In a recent in vitro study of the distribution of somatostatin receptors in cerebellar

Transient expression of somatostatin receptors 323

cells, Gonzalez et al.18 showed that somatostatin receptors are transiently expressed in immature neurons. Further histologic studies will be needed to precisely establish whether only neurons display somatostatin receptors during nervous system development. Also, because an in vitro study from Shinoda et ~1.~~ has demonstrated the expression of somatostatin-IR in astrocytes it should be kept in mind that non neuronal cells may express the peptide as well. However, so far, to our knowledge, all in v2’ro studies of the distribution of somatostatin-IR during neurogenesis looked upon the transient somatostatin-IR cells as neurons.

Neural tube

Between El 1.5 and E16.5, a widespread and transient increase of both somatostatin-R and somatostatin-IR levels was observed in the neural tube. The temporal pattern of expression of somatostatin-IR as observed herein is in agreement with a previous report of the distribution of somatostatin-IR in the rat developing spinal cord2’ but no data were available concerning the detection of somatostatin receptors in this structure at these early developmental stages. During the critical period of neurogenesis investigated, microscopic examination showed that some somatostatin receptors are associated with the developing white matter and premigratory immature cells of the VZ.

Around E13.5: intensely radiolabeled basal and intermediate plate areas may correspond to clusters of immature cells that also express glutamate decarboxylase (GAD).21.26 These immature neuronal pre- cursors are synthesizing GAD, prior to or during their terminal cell cycle, before they leave the VZ in their way of migration towards lateral regions of the neural tube. Interestingly, as observed here for the dynamic pattern of expression of somatostatin receptors, GAD-IR also appears to follow a ventral to dorsal gradient of expression between El 1-l6.25 Be- cause, on the one hand, somatostatin receptors seemed to be closely associated to the VZ and were detected following a ventrodorsal gradient, and on the other hand, the regression of the VZ adopt a similar pattern, it can reasonably be conceived that most of the somatostatin receptors visualized between El 1.5516.5 are associated with immature spinal neurons. This parallels earlier findings in the rat brain and visual system showing an association of somatostatin receptors with immature cells.J.5.‘7.‘8

We observed a transient expression of somatostatin receptors over the neural tube white matter and peripheral nerve during embryonic development. Since somatostatin-IR fibers were also seen in these structures together with somatostatin receptors (for example at E15.5 in the inferior funiculus), this suggests that somatostatin may also be involved in the maturational processes of spinal neuronal paths, as already evoked concerning the developing human pyramidal paths’ and rat optic pathways,4 respect-

ively. One may further speculate about the detection of low levels of somatostatin receptors in the ventral horns which are constituted by postmitotic neurons. If we assume that only immature neuronal somata display somatostatin receptors, this signal may orig- inate from scattered branches of the white matter tracts, In contrast, in the adult rat, neither ventral neurons nor the white matter of the spinal cord were shown to express somatostatin receptors.‘5~27~37

Unlike somatostatin receptors whose expression is mainly restricted to immature neurons (see above), somatostatin-IR was only observed in postmitotic neurons outside the VZ. For example, concerning the ventral horns of the neural tube at E12.5-13.5 (see Fig. 2A,B), it is apparent that areas with somatostatin-IR cell bodies (putative motoneurons) contain only low levels of somatostatin receptors. Besides, our results indicate that from El25 through E16.5, somatostatin-IR nerve fibers are present in neural tube areas containing somatostatin receptors. Some somatostatin-IR nerve fibers are present in neural tube areas containing somatostatin receptors. Some somatostatin-IR nerve fibers seen coursing through the developing gray matter, and particularly those more closely associated to the VZ, may bring somatostatin available to immature neurons. As previously hypothesized4,lC 18.20.21.29 somatostatin-IR nerve fibers might participate in controlling the proliferation, migration and/or the differentiation of immature cells as well as their incorporation into a specific neuronal circuitry.

Sensory derivatives

Between E11.5 and E16.5, a critical period of sensory neurogenesis. both somatostatin receptors and somatostatin-IR were widely and transiently associated with sensory derivatives.

In the DRG, somatostatin receptors and IR were concomitantly expressed and appeared associated with cell bodies around E13.5. Somatostatin receptors were first detected slightly later than somato- stdtin-IR and still visualized at E16.5 when somatostatin-IR had become undetectable in most of the sensory neurons. Such a delayed appearance of somatostatin receptors as compared to somatostatin- IR may depend on a lower sensitivity of the radioau- tography vs immunofluorescence. The asynchronic disappearance of both signals may result from the heterogeneous cellular content of the DRG. At least two classes of cells (large A and small B neurons) exist in the adult DRG. As previously noted for lumbar DRGs,~~ the time-interval in the generation of A and B neurons is similar to the time-lag between the disappcarancc of somatostatin-IR and somatostatin receptors. Furthermore, around E16.5, the radioautographic signal might correspond to intra- cellular somatostatin receptors-transport to distal central (neural tube through dorsal roots) or peripheral (peripheral nerves) sites. In contrast to what was observed during embryonic development, Manthy

324 E. Maubert et al.

ef ak2’ showed that adult DRG are devoid of somatostatin receptors.

Around E16.5, some scarce somatostatin-IR nerve fibers could be detected although no somatostatin-IR cell bodies were present in the DRG. These somatostatin-IR fibers, also described by Marti et at..28 may arise from the sympathetic neurons which are intensely somatostatin-IR at this stage (personal observations) and could reflect an early establishment of sympathetic-DRG pathways. Such an innervation was observed in the adult guinea-pig sensory ganglia.23

Thus, it appears that the transient detection of somatostatin receptors and -1R may occur concomitantly with the generation peak of sensory neurons and with the period during which these neurons establish connections with their target fields (for a review, see Ref. 29). Somatostatin receptors and -IR in sensory derivatives were localized in cells which had reached their final location in ganglia (i.e. after they had finished their migration along with other neural crest cells). Consequently, in contrast to the roles hypothetized for somatostatin in other CNS structures, in the DRG this peptide may not be involved in controlling the migration of sensory neurons.

Pitarmacoiogical characterization

Molecular cloning techniques have led to the iso- lation of cDNA encoding five different SSTR subtypes with specific pharmacological profile (for review see Ref. 10). At day E14.5, our preliminary pharmacological analysis suggests that, in the dorsal neural tube and DRG, the somatostatin receptors

which are transiently expressed may correspond to SSTRZ and/or SSTR4 subtypes(s). Recently, Wulfsen et a14’ have reported by semi quantitative polymerase chain reaction techniques that SSTRl and SSTR4 mRNA are more abundantly expressed than SSTRZ and SSTR3 mRNA during post natal development of the rat brain. However, selective patterns occurred depending on the brain structure:’ The pharmacological profile of the transiently expressed somatostatin receptors in the neural tube and sensory derivatives suggests that they belong to the SSTRZ/SSTR4 subtype(s). Such an hypothesis will have to be conforted by quantitative polymerase chain reaction analysis when available and compared with the pharmacological profile of other transiently expressed somatostatin receptor in the cerebellum’“” and visual system.+‘s5

CONCLUSION

This study extends the number of regions in which somatostatin and somatostatin receptors are transiently detected during neurogenesis. In addition, it shows that somatostatin-receptors, possibly of the SSTR2/SSTR4 subtype(s), are expressed earlier than previously shown and in a fashion congruent with the developmenta pattern of the neural tube and the sensory derivatives.

It appears that the transient expression of somatostatin receptors and/or somatostatin-IR characterizes critical episodes in the development of a cohort of neurons in the neural tube and the sensory derivatives. This unequivocally reinforces the notion that somatostatin has a fundamental role to play during neurogenesis in vertebrates.

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(Accepted 3 March 1994)

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Developmental patterns of somatostatin-receptors and somatostatin-immunoreactivity during early...

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