distribution of somatostatin-like immunoreactivity in the brain of the frog, rana esculenta, during...

Ž .Developmental Brain Research 106 1998 13–23

Research report

Distribution of somatostatin-like immunoreactivity in the brain of thefrog, Rana esculenta, during development

Mauro Vallarino a, Maura Mathieu a, Biagio D’Aniello b, Rakesh K. Rastogi b,)

a Istituto di Anatomia Comparata, UniÕersita di GenoÕa, Viale Benedetto XV 5, GenoÕa 16132, Italy`b Dipartimento di Zoologia, UniÕersita di Napoli, Via Mezzocannone 8, Napoli 80134, Italy`

Accepted 16 September 1997

Abstract

The anatomical distribution of somatostatin-like immunoreactivity in the central nervous system of the frog, Rana esculenta, duringdevelopment and in juvenile specimens was investigated by indirect immunofluorescence. Soon after hatching, at stages II-III,somatostatin-like immunoreactive structures were found in the preoptic-median eminence complex. In stage VI tadpoles, new groups ofimmunopositive perikarya and nerve fibers appeared in the diencephalon, within the ventral infundibular nucleus and in the ventral area ofthe thalamus, as well as in the medial pallium. In stages XII-XIV of development, immunopositive perikarya were also present in thedorsal infundibular nucleus of the hypothalamus and ventrolateral area of the thalamus. A small group of somatostatin-like immunoreac-tive neurons appeared in the posteroventral nucleus of the rhombencephalon. However, these neurons were not seen in later stages ofdevelopment. Tadpoles in stages XVIII, XXI-XXII and in juveniles were characterized by a wider distribution of immunoreactive cellbodies and fibers in the pallium. New groups of immunoreactive neurons were found in the dorsal and lateral pallium. The presence ofpositive perikarya in the lateral pallium is a transient expression found only in these stages. The organization of the somatostatinergicsystem was most complex during the metamorphic climax, with the appearance of positive cell bodies in the posterocentralis area of thethalamus, and in juvenile animals with the presence of perikarya in the ventral part of the medial pallium and within the central greyrhombencephali. In contrast to the adult frog, somatostatin neurons were not observed in the mesencephalon of tadpoles and juveniles.q 1998 Elsevier Science B.V.

Keywords: Somatostatin; Development; Brain; Frog; Immunohistochemistry

1. Introduction

Somatostatin-14 is a cyclic tetrapeptide, initially puri-fied from ovine hypothalamus and noted for its ability to

w xinhibit growth hormone release 4 . Subsequent studieshave shown that somatostatin-14 derives from a precursorŽ .prosomatostatin that generates, through proteolytic cleav-age, a 28-amino acid derivative called somatostatin-28,containing the sequence of somatostatin-14 at its C-

w xterminus 3,13,33 .In mammals, the neuroanatomical distribution of so-

matostatin-14 and somatostatin-28 has been studied exten-sively in the rat cen tral nervous systemw x15,17,20,25,30,31,35,36,45,51 . In mammals, these pep-tides have been shown to exert different central effects,including the inhibition of various hypothalamic neurose-

) Ž .Corresponding author. Fax: q39 81 7903342.

w xcretions 6,12,38 , regulation of striatal dopamine releasew x7 , and modulation of vagal and autonomic activitiesw x18,22,29,39 . In addition, somatostatin induces hyperther-

w xmia 5,47 , and modulates spinal sensitivity and reflexw xactivity 49 . The distribution of somatostatin-like material

has also been described in the brain of nonmammalianw xvertebrates, such as fishes 16,26–28,42 , amphibians

w x w x w x11,19,24,43 , reptiles 10,14,34,48 , and birds 1,11,37 . Incontrast, studies of developmental changes in brain so-matostatin-immunoreactive elements, which might give in-sight into its function, among vertebrates have been ex-tremely rare.

To our knowledge, within amphibia, there is no reporton the developmental aspects of somatostatin-immunoreac-tive neuronal system. The main objective of the presentinvestigation was, therefore, to describe the neuroanatomi-cal distribution of somatostatin in the brain of the frog,Rana esculenta, during its development.

0165-3806r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0165-3806 97 00162-4

( )M. Vallarino et al.rDeÕelopmental Brain Research 106 1998 13–2314

2. Materials and methods

2.1. Animals

In compliance with legislation on animal experiments,tadpoles in different stages of development after hatching,

Ž .and juveniles approximately 3-months old of R. escu-lenta, were sampled from the same pond. At least threeindividuals were used for each stage, and the developmen-tal stages were classified according to Tylor and Kollrosw x41 . All animals were anesthetized with tricaine methane-

Ž .sulfonate MS 222, Sigma Chemical Co. . The brain fromstages II-III, and successive larval stages and juvenileanimals, were rapidly dissected out and immersed in thefixative. All samples were fixed in freshly prepared Bouin’sfluid at room temperature under vacuum for 10 min,followed by immersion in fresh fixative for 4 h. Paraffin-embedded, 7-mm thick, serial transverse or sagittal sec-tions were mounted on chrome alumrgelatin-coated orpolylysine-coated glass slides.

2.2. Immunofluorescence procedure

Brain sections were rehydrated and processed for indi-rect immunofluorescence microscopy. Briefly, the sections

Žwere rinsed in cold phosphate-buffered saline PBS, 0.2 M,. Ž .pH 7.4 , preincubated with normal swine serum 1:50 for

20 min to reduce non specific staining, and incubated in adark moist chamber for 18 h at 48C with the somatostatin-14

Ž . Ž .antiserum Biomeda Corp. , or the somatostatin-28 1–14Ž .antiserum Cambridge Research Biomedicals diluted 1:200

in PBS. Rat pancreas sections were incubated with theŽ .somatostatin-28 1–14 antiserum as positive controls.

Then, the sections were rinsed several times in PBS andincubated for 1 h at room temperature with fluoresceinisothiocyanate-conjugated swine anti-rabbit gamma globu-

Ž .lin Dakopatts , diluted 1:100 in PBS. Finally, the sectionsŽ .were rinsed twice in PBS, mounted in glycerolrPBS 1:1 ,

and examined under a Zeiss epifluorescence microscope.Nomenclature of brain areas was based on the work of

w xWada et al. 46 .

Table 1Distribution of somatostatin-14-immunoreactive perikarya in the brain of the frog, R. esculenta, during development and in juvenile animals. Comparisonwith the distribution of somatostatin shown in adult brain of R. ridibundaa

Structure Stages Stage Stages Stage Stages Juvenile AdultaII-III VI XII-XIV XVIII XXI-XXII frog frog

Telencephalonolfactory bulb y y y y y y ypallium dorsalis y y y q q q qpallium mediale, pars dorsalis y q q q q q qpallium mediale, pars ventralis y y y y y q qpallium laterale, pars dorsalis y y y q q q ypallium laterale, pars ventralis y y y y y y ynucleus medialis septi y y y y y y qamygdala, pars lateralis y y y y y y qamygdala, pars medialis y y y y y y q

Diencephalonnucleus preopticus q q q q q q qarea ventralis anterior thalami y q y y y y qarea ventrolateralis thalami y y q q q q qarea ventromedialis thalami y y y y y y ynucleus dorsomedialis y y y q q q qanterior thalaminucleus dorsolateralis y y y y y y yanterior thalaminucleus posterocentralis thalami y y y y q q qnucleus posterolateralis thalami y y y y y y qnucleus entopeduncularis y y y y y y qnucleus infundibularis dorsalis y y q q q q qnucleus infundibularis ventralis y q q q q q q

Mesencephalonnucleus anteroventralis tegmenti mes. y y y y y y qnucleus profundus mesencephali y y y y y y qnucleus interpeducularis y y y y y y q

Rhombencephaloncentral gray rhombencephali y y y y y q qnucleus posteroventralis y y q y y y q

a w xq, Presence; y, absence; Laquerriere et al., 1989 24 .

( )M. Vallarino et al.rDeÕelopmental Brain Research 106 1998 13–23 15

2.3. Specificity of the immunoreaction

Preincubation of somatostatin-14 antiserum with 10y7

M synthetic somatostatin-14 resulted in complete loss ofthe immunoreaction. By contrast, preincubation of somato-statin-14 antiserum with 10y7 M synthetic somatostatin-28did not change the intensity of the immunostaining. Noimmunostaining was observed when the somatostatin-14antiserum was replaced by nonimmune rabbit serum orPBS. Incubation of rat pancreas sections with somato-statin-28 antiserum revealed the presence of positive cellsin the islets of Langerhans.

3. Results

The distribution of somatostatin-like immunoreactivecell bodies and fibers in the brain has been investigated inanimals whose age varied from early developmental stagesup to the juvenile stage. Incubation of sections with anti-somatostatin-14 revealed the presence of distinct popula-tions of cell bodies in all stages investigated. In contrast,no immunoreactive perikarya and nerve fibers were ob-served using the antiserum raised against somatostatin-28.The anatomical distribution of somatostatin-14 immunore-active perikarya and fibers in the brain of Rana esculenta

Žduring the larval stages stages: II-III, VI, XII-XIV, XVIII,.XXI-XXII and in juvenile animals is shown in Table 1

and schematically illustrated in Fig. 1.

3.1. LarÕal deÕelopment

3.1.1. Stages II-IIISoon after hatching, a small number of somatostatin-like

immunoreactive neurons was present in the hypothalamus,at the level of the medial and caudal regions of the

Ž .preoptic nucleus nucleus preopticus . These elements werelocated in the intermediate layers of the nucleus and

Ž .showed a weak immunofluorescence Fig. 2 . Althoughfiber projections of these neurons were hardly discerniblein the hypothalamus, a few weakly-immunofluorescentfibers were observed in the mediane eminence. Duringthese early stages of development, the other regions of thebrain did not show any somatostatin-like immunoreactiv-ity.

3.1.2. Stage VIIn the brain of stage VI tadpoles, the relative frequency

and topography of somatostatin-immunoreactive elementsshowed remarkable changes. In particular, in the hypo-thalamus, somatostatin-like immunoreactive perikarya were

Ž .found in the medial part of the preoptic nucleus Fig. 3 , asreported in stages II-III, as well as in the rostral part of the

Žventral infundibular nucleus nucleus infundibularis ven-. Ž .tralis Fig. 4 . During this stage, furthermore, somato-

statin-like immunoreactive perikarya appeared scattered in

Fig. 1. Schematic camera lucida reconstruction of sagittal sections throughthe brain of the frog, Rana esculenta, during development. Distribution

Ž .of somatostatin-14-immunoreactive perikarya triangles and nerve fibersŽ . Ž . Ž . Ž .dots is shown. A stages II-III; B stage VI; C stages XII and XIV;Ž .D juvenile. 1, nucleus preopticus; 2, nucleus infundibularis ventralis; 3,pallium mediale, pars dorsalis; 4, area ventralis anterior thalami; 5,nucleus infundibularis dorsalis; 6, nucleus posteroventralis rhomben-cephali; 7, pallium dorsalis; 8, pallium laterale, pars dorsalis; 9, nucleusdorsomedialis anterior thalami; 10, central grey rhombencephali. Scalebars1 mm.

the telencephalon, at the level of the dorsal extension ofŽ . Ž .the medial pallium pallium mediale, pars dorsalis Fig. 5

Žand in the anteroventral area of the thalamus area ven-.tralis anterior thalami . Immunostained nerve fibers were

spread in the telencephalon within the medial pallium, inthe infundibular region of the hypothalamus, as well as in

Ž .the median eminence Fig. 6 .

3.1.3. Stages XII-XIVIn stages XII-XIV of larval development, a substantially

larger number of immunoreactive cell bodies and addi-tional groups of immunostained perikarya were found inthe brain of R. esculenta. Similar to stage VI, severalsomatostatin-like immunoreactive perikarya were found inthe dorsal component of the medial pallium, the medialregion of the preoptic nucleus, and the rostral part of the

Ž .ventral infundibular nucleus Fig. 7 . During these devel-opmental stages, a large number of bright somatostatin-im-


ŽFig. 2. Transverse section of stages II tadpole brain showing the presence of a few weakly-stained somatostatin-like immunoreactive perikarya indicated. Ž .by white triangles in the caudal region of the preoptic nucleus. III, third ventricle preoptic recess . Scale bars100 mm.

Fig. 3. Stage VI tadpole brain with white triangles pointing to the somatostatin-like immunoreactive cell bodies in the medial preoptic nucleus. Transversesection. Scale bars100 mm.

Fig. 4. Stage VI tadpole. Transverse section through the rostral part of the ventral infundibular nucleus showing the location of somatostatin-containingŽ .neurons triangles . Scale bars100 mm.

Ž .Fig. 5. Stage VI tadpole. Somatostatin-like immunoreactive cell body triangle and nerve fibers through the dorsal part of the medial pallium. Transversesection. Scale bars100 mm.

munoreactive perikarya appeared in the rostral part of theŽdorsal infundibular nucleus nucleus infundibularis dor-

. Ž .salis Fig. 8 and within the anterior region of the ventro-Ž .lateral area of the thalamus area ventromedialis thalami

Ž .Fig. 9 . No somatostatin-like positive perikarya were pre-sent in the medial and caudal regions of the dorsal andventral infundibular nuclei. More caudally, a new group ofimmunoreactive cell bodies appeared in the brainstem, at

Ž .Fig. 6. Stage VI tadpole. Somatostatin-like immunofluorescent nerve fibers triangles in the median eminence. Transverse section. Scale bars100 mm.

Fig. 7. Stage XIV tadpole. Transverse section at level of the rostral part of the ventral infundibular nucleus, showing the presence of numerousŽ .somatostatin-like immunoreactive neurons triangles . Scale bars100 mm.

Fig. 8. Stage XIV tadpole. Transverse section showing several bright somatostatin-like immunofluorescent neurons, indicated by rows of white triangles, inthe rostral part of the dorsal infundibular nucleus. IR, infundibular recess. Scale bars100 mm.

Fig. 9. Stages XIV tadpole. Transverse section through the anterior region of the ventrolateral area of the thalamus, showing the presence of a group ofbright somatostatin-immunofluorescent perikarya. Scale bars100 mm.


Žthe level of the posteroventral nucleus nucleus pos-. Ž .teroventralis of the rhombencephalon Fig. 10 . Numerous

immunostained nerve fibers were seen coursing throughthe ventral part of the infundibular region of the hypothala-

Ž .mus, median eminence Fig. 11 , medial pallium, thala-mus, and in the caudal rhombencephalon.

3.1.4. Stage XVIIIAt stage XVIII, the telencephalon exhibited, in addition

to somatostatin-like immunoreactive perikarya in the dor-sal part of the medial pallium, a large number of positive

Ž .neurons both in the dorsal pallium pallium dorsalis andŽwithin the dorsal component of the lateral pallium pallium

. Ž .laterale, pars dorsalis Fig. 12 . Like in the early stages ofdevelopment, no somatostatin immunoreactivity was foundin the olfactory bulbs. In the preoptic nucleus, the presenceof somatostatin-like immunoreactivity was observed bothin the medial and caudal parts of the nucleus. In theseregions the somatostatin-immunoreactive neurons werecomparatively more frequent than in the brain of earlierstages of development, both in the periventricular and

Ž .lateral layers Fig. 13 . During this stage, in addition tosomatostatin-like immunoreactive perikarya in the anteriorregion of the ventrolateral area of the thalamus, a newgroup of positive cell bodies was observed through the

Žanterior dorsomedial nucleus of the thalamus nucleus.dorsomedialis anterior thalami . In the infundibular nuclei,

the distribution of somatostatin was very similar to thatdescribed in stages XII-XIV. Also in this stage, somato-statin-like immunoreactive nerve fibers were mainly con-centrated throughout the pallium, infundibulum, medianeminence, and thalamus. No immunostained structures wereobserved in the brainstem.

3.2. Metamorphic climax

3.2.1. Stages XXI-XXIIDuring the metamorphic climax, in the telencephalon

the distribution of somatostatin-immunoreactive cell bod-ies and nerve fibers was very similar to that described instage XVIII. A large number of immunoreactive perikaryawas observed in the medial and caudal parts of the preop-tic nucleus. However, during these stages, a new subset ofimmunoreactive cell bodies appeared in the rostral compo-

Ž .nent of the preoptic nucleus Fig. 14 . In the infundibularregion most somatostatin-immunoreactive cell bodies wereconcentrated in the rostral part of the ventral infundibular

Ž .nucleus Fig. 15 . In the thalamus, like in stage XVIII, wefound several immunoreactive cell bodies within the ante-rior dorsomedial nucleus, and in the rostral and caudalparts of the ventrolateral area. In this region, a new groupof immunostained cell bodies appeared in the rostral re-

Žgion of the posterocentrale nucleus nucleus posterocen-.tralis thalami . These elements were mainly located in the

medial and lateral parts of the nucleus and some of themŽ .showed a weak immunostaining Fig. 16 . Other areas of

the brain did not show any somatostatin-immunoreactivestructures. The distribution of positive fibers was similar tothat reported in stage XVIII.

3.3. JuÕenile animals

In juvenile animals, the neuroanatomical organizationof somatostatin-like immunoreactive perikarya and nervefibers showed some changes as compared to that describedduring the metamorphic climax. Three new subsets ofimmunoreactive cell bodies were observed. One group ofperikarya was found in the telencephalon, along the ventral

Žportion of the medial pallium pallium mediale, pars ven-.tralis . A second group of immunoreactive cell bodies was

observed within the medial part of the ventral infundibularŽ .nucleus Fig. 17 . More caudally, a third group of positive

neurons, weakly immunostained, was identified in thebrainstem within the central gray of the rhombencephalonŽ .central gray rhombencephali . In all other regions of thebrain, the distribution of immunoreactive perikarya wasvery similar to that described in the brain of stages XXI-XXII. Immunoreactive fibers were found in the hypothala-mus, pallium, thalamus, and through the rhombencephalon.

Immunopositive cell bodies were mostly spherical oroval in shape. In the infundibular region, however, they

Ž .were rather elongated and fusiform Figs. 7, 14 and 17 .The intracellular localization of immunofluorescence wasconfined to the cytoplasm of the cell body. Most of theneurons appeared to be unipolar with a club-like processemerging from the cell body. Bipolar neurons were fre-quently observed in the infundibular nuclei. Although someimmunopositive neurons are located in the vicinity of the

Ž .Fig. 10. Stage XIV tadpole. Somatostatin-like immunoreactive neurons triangles in the posteroventral nucleus of the rhombencephalon. Transversesection. Scale bars100 mm.

Fig. 11. Stage XIV tadpole. This figure shows the presence of numerous somatostatin-like immunoreactive nerve fibers in the median eminence. Sagittalsection. Scale bars100 mm.

Ž .Fig. 12. Stage XVIII tadpole. Somatostatin-immunofluorescent neurons triangles in the dorsal part of the lateral pallium. Transverse section. Scalebars100 mm.

Fig. 13. Stage XVIII tadpole. White triangles pointing to the somatostatin-like immunostained perikarya in the dorsal part of the medial preoptic nucleus.Transverse section. Scale bars100 mm.


cerebral ventricles, particularly those in the preoptic andŽ .infundibular nuclei Figs. 2, 3, 14, 15 and 17 , we have no

clear-cut indication of immunopositive processes in con-tact with the cerebrospinal fluid at any point of develop-ment.

4. Discussion

Developmental changes in the immunohistochemicaldistribution of somatostatin-like substances in the brain ofa representative amphibian, the frog, Rana esculenta, havebeen described for the first time. These results indicate thatthe brain of R. esculenta, during development and up to

Ž .three months after metamorphosis juvenile , contains apeptide with immunological properties related to somato-statin-14 but not to the N-terminal region of somatostatin-28. Consistent with this observation, previous studies haveshown that the sequence of somatostatin-14 is identical inall vertebrates so far investigated, whereas the sequence of

Ž .somatostatin-28 1–12 in adult frog exhibits two substitu-tions as compared to the corresponding mammalian se-

w xquence 40 . In addition, the adult frog brain contains aŽsecond form of somatostatin-14 with two substitutions Pro

2 13.for Gly , and Met for Ser compared with mammalianw xsomatostatin-14 44 . Concerning this second molecular

form, we can not exclude its possible existence also in thebrain of frog during development that might not have beendetected with our antisera. However, it is also possible thatour somatostatin antisera might have detected both somato-statin-14 isoforms. Further studies are required to clarifythis issue.

Our results show that the distribution of somatostatin-immunoreactive structures in the brain of the frog changesduring development. Soon after hatching, in stages II-III,somatostatin-containing neurons were confined to the pre-optic nucleus. During the successive developmental stages,the frequency of somatostatin-immunoreactive perikarya inthe preoptic nucleus increased progressively. Theseperikarya were particularly abundant in the developmentalstages XVIII and XXI-XXII, and in juveniles. These ob-servations, together with the finding of immunoreactivefibers within the median eminence, suggest that somato-

statin may act as a hypophysiotropic factor already at anearly stage of development. The high concentration ofsomatostatin immunoreactivity in the median eminencesuggests that somatostatin, in addition to its inhibitory

w xaction on growth hormone secretion 4 , might also regu-late the release of other pituitary hormones. This hypothe-sis finds support from previous studies showing that so-matostatin inhibits prolactin secretion in the teleost fishw x w x16,50 , stimulates ACTH release in the trout 23 , and

w xmodulates FSH secretion in the rat 21 . It is noteworthythat FSH and ACTH are expressed in the pituitary of

w xtadpoles very early during development 8,32 . The pres-ence of somatostatin immunoreactivity in the preopticarea-median eminence complex of tadpoles is consistentwith previous results reported in the adult Rana tempo-

w x w x w xraria 43 , Rana catesbeiana 19 and Rana ridibunda 24Ž .see Table 1 . The appearance of immunopositive cellbodies and fibers in the ventral component of the in-fundibular nucleus of the hypothalamus and within thetelencephalon at the level of the dorsal part of the medialpallium is a common feature of the successive stages of

Ž .development as well as of the adult frog see Table 1w x19,24 . The location of somatostatin-14 in the medialpallium suggests that, from developmental stage VI on-ward, somatostatin may act in tadpole as a neuromodulatorof several sensory informations, including olfactory, vis-ceral and somatosensory stimuli, relayed from the dorsalthalamus, dorsal and lateral pallia, subpallium, and theolfactory bulb. The finding of somatostatin-like cell bodiesand fibers in the medial pallium is consistent with thelocation of positive neurons in the same region of other

w x w xland vertebrates, including reptiles 34 , birds 1 and mam-w xmals 35,36,45,51 .

In the brain of stages XII-XIV tadpoles, two newgroups of somatostatin-immunoreactive cell bodies weredetected within the diencephalon: one at the level of thedorsal infundibulum, and another in the ventrolateral areaof the thalamus. More caudally, an additional subset ofsomatostatin-immunoreactive neurons was found in theposteroventral nucleus of the rhombencephalon. Whereasthe presence of somatostatin-immunoreactive neuronswithin the two diencephalic nuclei also characterized thesuccessive developmental stages, the location of somato-

Ž .Fig. 14. Metamorphic climax stage XXI . White triangles indicate somatostatin-containing neurons in the rostral part of the preoptic nucleus. Transversesection. Scale bars100 mm.

Ž . Ž .Fig. 15. Metamorphic climax stage XXII . Transverse section showing the location of somatostatin-like immunofluorescent neurons triangles in therostral part of the ventral infundibular nucleus. Scale bars100 mm.

Ž . Ž .Fig. 16. Metamorphic climax stage XXII . Somatostatin immunoreactive parikarya white trinagles in the rostral region of the posterocentral nucleus ofthe thalamus. Transverse section. Scale bars100 mm.

Fig. 17. Juvenile. Somatostatin-like immunoreactive cell bodies and nerve fibers in the medial part of the ventral infundibular nucleus. POR, postopticrecess. Transverse section. Scale bars100 mm.


statinergic neurons in the posteroventral nucleus of therhombencephalon appears to be a transient expression ofstages XII-XIV. Advanced larval stages and juvenile ani-mals did not show any immunoreactive perikarya in thisnucleus. However, somatostatin-containing neurons weredescribed in the posteroventral nucleus of the rhomben-

Ž .cephalon in the adult frog, Rana ridibunda see Table 1w x24 . The presence of somatostatin-like immunoreactiveperikarya in the diencephalon of tadpoles agrees with thelocation of dense populations of cell bodies in the preopticarea and hypothalamus of other vertebrates, including fishesw x w x w x16,26–28 , reptiles 2,10,14,48 , birds 37 , and mammalsŽ w x .see 9 , for review . However, the presence of somato-statin neurons in the thalamus has been reported only in

Ž w x.the frog see 24 . Stage XVIII of larval development ischaracterized by the appearance of somatostatin-like im-munoreactive cell bodies both in the dorsal pallium andlateral pallium. The dorsal pallium relays visual input tothe medial pallium, and receives olfactory bulb projec-tions. The lateral pallium, in addition to olfactory informa-tions, receives afferent projections from several brain re-gions, including the medial pallium, septum, amygdala,preoptic area, thalamus, and the infundibular hypothala-mus. Thus, beginning this developmental stage onward,somatostatin could play an important role as neuromodula-tor in the pallium. The presence of somatostatin-im-munoreactive neurons in the lateral pallium is a transientexpression of larval stages XVIII and XXI-XXII and ofjuvenile animals. By contrast, in this region no im-munopositive cell bodies have been described in the adult

w xbrain 24 . During stages XXI-XXII, the organization ofthe somatostatinergic system only changed in the dien-cephalon in which a new group of neurons appeared in theposterocentral area of the thalamus, whereas in juvenileanimals, new somatostatin positive cell bodies were foundin the ventral part of the medial pallium. Our results showthat somatostatinergic elements are more widespread in thebrain of juvenile animals than in that of any of the larvalstage investigated. However, if we compare the distribu-tion of somatostatin in juvenile animals with the distribu-tion reported in the adult frog, in the latter new groups ofpositive cell bodies have been reported in the telen-

Ž .cephalon medial septal region and amygdala , in theŽdiencephalon area ventralis anterior thalami, nucleus pos-

.terolateralis thalami, nucleus entopeduncularis and in sev-Žeral regions of the mesencephalon nucleus anteroventralis

tegmenti mesencephali, nucleus profundus mesencephali.and nucleus interpeduncularis and rhombencephalon

Ž .central gray rhombencephali and nucleus posteroventralisw x24 . Thus, the distribution of the somatostatinergic systemprogressively increases during larval development as wellas during the transition from the juvenile to the adult frog.This fact points all too well to the possibility that somato-statin-like peptides may act as central neuromodulators ofprogressively increasing number of behavioral and physio-logical activities, from tadpoles through adulthood.

Acknowledgements

This study was supported by grants from the Universi-Ž .ties of Genova and Naples Federico II Italy , and

M.U.R.S.T., Italy.

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distribution of somatostatin-like immunoreactivity in the brain of the frog, rana esculenta, during...

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