www.elsevier.com/locate/jchemneu

Journal of Chemical Neuroanatomy 32 (2006) 127–142

Proliferative activity in the frog brain:

A PCNA-immunohistochemistry analysis

Franca Raucci a, Maria M. Di Fiore a, Claudia Pinelli a, Biagio D’Aniello b,Luciano Luongo b, Gianluca Polese b, Rakesh K. Rastogi b,*

a Department of Life Sciences, Second University of Naples, 81100 Caserta, Italyb Department of Structural and Functional Biology, University of Naples Federico II, Via Cinthia, MSA Campus, 80126 Naples, Italy

Received 12 April 2006; received in revised form 12 July 2006; accepted 5 August 2006

Available online 20 September 2006

Abstract

By means proliferating cell nuclear antigen (PCNA) immunohistochemistry, we have provided a detailed neuroanatomical mapping of

proliferative activity during development and adulthood in the frog (Rana esculenta) brain. Western blot analysis confirmed the presence of this

protein in brain extracts from adults and tadpoles. Proliferative activity was observed in the ventricular and subventricular zones throughout the

brain. The present study provides details as to which of the morphologically distinguishable brain region(s) has a long-lasting proliferative activity

and in which region this activity undergoes a progressive decrease during development. In the subventricular zones of the third ventricle, PCNA-

labeled cells were particularly abundant in the magnocellular preoptic nucleus and the ventromedial thalamic nucleus. It was observed that

proliferation zones are present practically in all major subdivisions of the forebrain, midbrain and hindbrain, including the cerebellum in which

PCNA-labeled cells were located in the outer granular layer and the inner molecular layer. The habenulae, epiphysis and isthmic nuclei never

showed the presence of PCNA-immunoreactive nuclei. The widespread proliferative activity implies that the frog brain has a great potential for

neurogenesis/gliogenesis not only during larval development but also in the adulthood.

# 2006 Elsevier B.V. All rights reserved.

Keywords: PCNA; Frog brain; Cell proliferation; Immunohistochemistry; Development

1. Introduction

The proliferating cells confer to the brain the capacity of

neurogenesis/gliogenesis and regeneration. Evidence accumu-

lated for over 30 years indicates that during adulthood, all

vertebrates possess potential to produce new neurons/astro-

cytes/glial cells in the central nervous system; this potential has

been observed within the framework of a seasonal or a

hormone-regulated behavioural rhythm, or in the replacement

of the damaged neurons by newly generated ones after

experimentally induced injuries (Nottebohm, 1985; Alvarez-

Buylla, 1990; Lois and Alvarez-Buylla, 1993; Zupanc and

Horschke, 1995; Perez-Canellas and Garcıa-Verdugo, 1996;

Chiasson et al., 1999; Dawley et al., 2000; Ino and Chiba, 2000;

Margotta et al., 2000, 2005; Ekstrom et al., 2001; Zupanc,

2001; Garcia-Verdugo et al., 2002; Alvarez-Buylla et al., 2002;

* Corresponding author. Tel.: +39 081 679209; fax: +39 081 679233.

E-mail address: rastogi@unina.it (R.K. Rastogi).

0891-0618/$ – see front matter # 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.jchemneu.2006.08.001

Rodrıguez-Perez et al., 2003; Rankin et al., 2004; Romero-

Aleman et al., 2004; Vidal Pizarro et al., 2004; Dewulf and

Bottjer, 2005). These studies have shown that neurogenesis/

gliogenesis occurs in the brain of adult teleost fishes,

amphibians, reptiles, birds and mammals. A neuroanatomically

more widespread potential for cell proliferation in the adult

brain has been suggested in some anamniote vertebrates, such

as teleost fishes (Ekstrom et al., 2001; Zupanc, 1999) and

amphibians (Richter and Kranz, 1981; Bernocchi et al., 1990;

Dawley et al., 2000; Margotta et al., 2000). In the adult

mammals, including humans, neurogenesis/gliogenesis has

been described to occur in a few circumscribed areas, viz.,

olfactory bulbs, hippocampus and cortex, although it is debated

that adult neurogenesis is far more widespread than these

studies have described (see Eriksson et al., 1988; Gould et al.,

1999; Rietze et al., 2000; Garcia-Verdugo et al., 2002;

Rodrıguez-Perez et al., 2003; Bedard and Parent, 2004).

Within the wide panorama of such studies in vertebrates,

amphibian models (urodeles and anurans) have had a rather

conspicuous role. While many authors focused their attention

F. Raucci et al. / Journal of Chemical Neuroanatomy 32 (2006) 127–142128

on the process of regeneration in the central nervous system

following lesions or thermal shocks (Minelli et al., 1982; Del

Grande et al., 1984, 1990; Franceschini et al., 1992; Benraiss

et al., 1999; Yoshino and Tochinai, 2004), several others

investigated upon the development/neurogenesis of specific

parts of brain such as the olfactory bulbs (Burd and Sein,

1998), vocal pathway (Gorlick and Kelley, 1987), cerebellum

(Filoni and Margotta, 1971; Gona, 1976; Uray et al., 1987,

1988), isthmic nucleus (Tay and Straznicky, 1980; Dann and

Beazley, 1989), optic tectum (Straznicky and Gaze, 1972;

Reznikov and Maliovanova, 1979; Dann and Beazley, 1988),

hypothalamus (Chetverukhin and Polenov, 1993; Polenov and

Chetverukhin, 1993), preoptic recess (Onishchenko et al.,

1983), telencephalon (Margotta et al., 1999b, 2000) or of the

entire brain (Bernocchi et al., 1990; Dawley et al., 2000;

Wullimann et al., 2005). These studies have demonstrated

that in the adult frogs, newts and salamanders the brain has

the potential for cell proliferation in certain localized areas

under normal and experimental conditions. It is surprising,

however, that a detailed neuroanatomical mapping of the

proliferative activity in the adult brain as well as during

development is not available for any single species. Regarding

the developmental changes in the proliferative activity within

the vertebrate brain there are only a few studies available:

cyclostomes (Villar-Cheda et al., 2002, 2005a,b), teleost fishes

(Wullimann and Puelles, 1999; Wullimann and Knipp, 2000;

Wullimann and Mueller, 2004), alligator hindbrain (Pritz,

2003) and mouse (Martinez and Puelles, 2000). Only recently

has data on the developmental pattern of the neurogenic and

proneural gene expression as well as a gross picture of the

proliferative activity in the brain of an amphibian, the African

clawed frog (Xenopus laevis), been published (Wullimann

et al., 2005). A more recent study has described the

proliferation pattern in the auditory medulla of the bullfrog

during development (Chapman et al., 2006).

Although cell proliferation in the nervous system of

vertebrates has been widely investigated by tritiated thymidine

labeling and 5-bromo-20-deoxyuridine (BrdU) immunocyto-

chemistry, a high degree of reliability consistently emphasized

for the more recent proliferating cell nuclear antigen (PCNA, an

auxillary protein to DNA-polymerase delta) has made of it the

cell cycle marker protein of choice (Rankin et al., 2004; Valero

et al., 2005). PCNA is a nuclear protein that is synthesized in the

G1 and S phases of the cell cycle and is, therefore, correlated

with the cell proliferative stage (Jaskulski et al., 1988;

Tsurimoto, 1999; Wullimann and Puelles, 1999; Rankin

et al., 2004). Additionally, PCNA analysis of cell proliferation

in the vertebrate nervous system has been found to be

comparable to the analysis using tritiated thymidine (see

Ramirez et al., 1997; Ekstrom et al., 2001). PCNA labeling has

been described in the ventricular and subventricular zones

(described as ependymal/subependymal cell areas in the adult

brain, and matrix areas in general by some other authors) of the

vertebrate brain (see Margotta and Morelli, 1996; Ino and

Chiba, 2000; Margotta et al., 1999a; Wullimann and Puelles,

1999; Wullimann et al., 2005). Thus, immunohistochemistry

for PCNA was our instrument for the present study to delineate

the developmental and adult profile of the proliferative activity

in the brain of a ranid frog, Rana esculenta.

2. Materials and methods

2.1. Animals

Tadpoles and adults of R. esculenta were collected from a pond, nearly

75 km northeast of Naples, at an altitude of 950 m ca. Tadpoles were identified

according to the staging table by Gosner (1960) and were divided into six

developmental groups as follows: stages 24–25 (hindlimb buds not yet visible)

as Group I, stages 27–28 (limb buds are visible) as Group II, stages 36–37 (toe

development) as Group III, stage 40 as Group IV, stage 42 (forelimbs emerged)

as Group V and stages 44–45 (tail resorption; metamorphic climax) as Group

VI. For immunocytochemical studies, 8–10 specimens were used for each

developmental group; only 6 adults (collected in the month of June), 3 males

and 3 females were used for this study, all in the weight range of 14 � 1 g.

Immediately after their collection, all samples were anesthetized with MS222

(tricaine methanesulfonate, Sigma Chemical Co.). Adult animals were perfused

intracardially first with cold PBS (pH 7.4) followed by Bouin’s fluid and the

brain was rapidly dissected out and immersed in Bouin’s fluid for 24 h at field/

room temperature. In tadpoles from Groups I to IV, the head was fixed in toto. In

more advanced stages of development, the brain was surgically removed from

the brain case and immersed in the fixative. Bouin-fixed material was then

dehydrated in ethanol, cleared in Bioclear, and embedded in paraffin. Sections

were cut at 5–7 mm in the sagittal or transverse plane and serially placed on

poly-L-lysin coated glass slides.

2.2. Immunohistochemistry

The paraffin sections were dewaxed in Bioclear, rehydrated in a decreasing

ethanol series, rinsed in deionized distilled water and PCNA-immunostained as

described. Briefly, the endogenous peroxidases were suppressed by incubation

of sections in 0.1% sodium azide with 0.3% hydrogen peroxide for 30 min at

room temperature. The sections were then incubated in normal goat serum

(1:20; Sigma Chemical Co., Italy) in 0.1 M phosphate buffer saline (PBS; pH

7.4) for 45 min at room temperature to reduce undesired background staining.

Intracerebral localization of PCNA was achieved by sequentially applying on

brain sections a monoclonal mouse anti-PCNA (1:10,000; Dako Corp., Den-

mark) in 10% bovine serum albumin (BSA) overnight at 4 8C in a dark moist

chamber, biotinylated secondary antibody (goat anti-mouse IgG diluted 1:500;

Sigma) for 45 min, and peroxidase-coupled streptavidin (1:200; Pierce, IL,

USA) for 1 h at room temperature. Then 3% DAB (3.30-diaminobenzidine

tetrahydrochloride; Sigma) with 0.03% hydrogen peroxide in Tris buffer

(0.05 M, pH 7.6) was used as chromogen. After each reaction step and after

DAB-peroxidase visualization, sections were washed thoroughly in PBS (two

changes of 10 min each). Sections were then dehydrated through a series of

graded alcohols, cleared in Bioclear, and coverslipped in a synthetic medium.

Control tests were done by: (a) omission of the primary antibody or (b)

substitution of the primary antiserum with PBS or normal goat serum

(1:500; Dako) in 10% BSA; no immunostaining was observed.

All experimental procedures were in line with the European Community’s

Council Directive (86/609/EEC).

2.3. Western blot analysis

Brains from tadpoles belonging to the developmental Groups III–VI, and

adults (male and female) were surgically removed under MS222 anaesthesia,

rapidly frozen in liquid nitrogen and stored at �80 8C. Frozen brains were

homogenized directly in lysis buffer containing 5 mM Tris–HCl, 150 mM

NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 1 mM

phenylmethylsulfonyl fluoride (PMSF), 1 mg leupteptin, 0.5 mM sodium ortho-

vanadate, 20 mM sodium pyrophosphate and clarified by centrifugation at

14,000 � g for 10 min. Protein concentration was estimated using a modified

Bradford assay (Bio-Rad, Hercules, CA). Electrophoresis was performed on

SDS-PAGE by loading 30 mg proteins for each sample and proteins electro-

transferred onto nitrocellulose membrane (Immobilion Millipore Corporation,

F. Raucci et al. / Journal of Chemical Neuroanatomy 32 (2006) 127–142 129

Bedford, MA); completeness of transfer was assessed using prestained protein

standards (Bio-Rad). Nitrocellulose was treated for 2 h with a blocking solution

(5% non-fat powdered milk in 25 mM Tris, pH 7.4; 200 mM NaCl; 0.5% Triton

X-100) and incubated overnight with mouse monoclonal antibody against

recombinant PCNA, diluted 1:2000. After buffer washing the membranes were

incubated for 60 min with peroxidase-conjugated anti-mouse secondary anti-

body (1:5000; Chemicon, CA). Immunoreactivity was detected by enhanced

chemiluminescence system (Amersham Life Science, UK). The optical density

of the bands was obtained by densitometry and normalized using a SCAN

software.

Data were compared by analysis of variance followed by Duncan’s test for

multi-group comparison and the Student’s t-test for between-group comparison.

All data were expressed as mean � standard deviations. The level of signifi-

cance was taken at P < 0.01.

2.4. Photodocumentation

Digital photomicrographs were made with a Canon Digital camera on a

DMBR Leica photomicroscope. The images obtained were slightly adjusted for

brightness and contrast with Adobe Photoshop 6.0. Photomontages and lettering

were done using CorelDraw 9.0. Western blotting chart was drawn using

PowerPoint.

3. Results

3.1. Immunohistochemistry

The following description has been arranged in progressive

stages of development and adult.

3.1.1. Group I (stages 24–25)

Immediately prior to the appearance of limb buds, the

majority of cell nuclei in the developing brain were

Fig. 1. Photomicrograph of a medial sagittal section of the brain of a newly hatc

proliferative activity in the periventricular area as indicated by the abundance of PCN

distribution of PCNA-ir nuclei (arrows) in the telencephalon (A) and thalamic area (B

abbreviations as in Fig. 5. Scale bars: (inset) 100 mm; (A and B) 50 mm.

immunolabeled with the PCNA antibody (Fig. 1A and B).

All cells of the ventricular zone and the majority of those

located in the subventricular zone were strongly immunos-

tained. The brain ventricles were virtually lined with PCNA-

immunostained cells throughout the rostrocaudal axis (see inset

of Fig. 1). In other words, immunostaining occurred

uninterrupted throughout the ventricular lining, whereas more

distant cells showed decreasing intensity of immunoreactivity

and many were totally unstained. The greatest accumulation of

immunoreactive cells was seen in the pseudostratified tectum

mesencephali (optic tectum); all cells of the tectum were

immunostained with anti-PCNA.

3.1.2. Group II (stages 27–28)

As compared with the earlier group of tadpoles, the brain of

these tadpoles had undergone changes to the extent that it was

possible to make out the gross neuroanatomical limits of the

forebrain, midbrain and hindbrain regions, as well as the brain

ventricles, the developing cerebellum and the infundibular

stalk. In these tadpoles the proliferative zones of the brain could

be distinguished regionally. As yet, the PCNA-immunostained

cell lining was several layers thick in all brain ventricles (see

Fig. 2). In the telencephalon, through its entire length, the

lateral ventricles were lined with several layers (ventricular and

subventricular zones) of ir cells (Fig. 2A), indicative of a high

proliferative activity. Similarly, in the most dorsal, medial and

lateral parts of the diencephalon, several layers of ir cells lined

the third ventricle (Fig. 2B). The preoptic recess was easily

discernible (not seen in the section in Fig. 2) and was

surrounded by proliferating cells distributed in the ventricular

hed tadpole (prior to hindlimb bud; Group I) showing the occurrence of high

A-ir nuclei (arrows). Enlargements of the boxed areas in the inset showing the

). Empty triangles indicate some radially displaced ir nuclei. TH, thalamus; other

F. Raucci et al. / Journal of Chemical Neuroanatomy 32 (2006) 127–142130

Fig. 2. Photomicrograph of a sagittal section (inset) of a hindlimb bud stage tadpole (Group II). Enlargements of the boxed areas in the inset, showing the distribution

of PCNA-ir nuclei (arrows) in the telencephalon (A), thalamic area (B), infundibular hypothalamus (C), optic tectum (D), cerebellum (E) and rhombencephalon (F).

Note the total absence of labeled nuclei within the habenula (B). Abbreviations as in Fig. 5. Scale bars: (inset) 500 mm; (A and C–F) 100 mm; (B) 100 mm.

and subventricular zones. Similarly, the infundibular recess, in

its early formation, was surrounded by a mass of PCNA-ir cells

(Fig. 2C). The optic tectum appeared very similar to that

observed in the earlier group and the entire pseudostratified

mass of cells was strongly immunostained (Fig. 2D). The

cerebellum appeared quite well developed and in any section

along its entire width most of it was composed of PCNA-

labeled cells (Fig. 2E). Curiously, immediately underneath the

tip of the cerebellar plate there was an area of unlabeled cells.

The proliferative activity was very pronounced also at the level

of the rhombencephalon, around the fourth ventricle (Fig. 2F).

3.1.3. Group III (stages 36–37)

In tadpoles with growing hindlimbs, the lateral ventricles

were lined with a few layers of ir cells as in the earlier group (see

inset of Fig. 3); a comparatively similar condition was also

observed for the periventricular cell linings in the upper half of

the third ventricle with the habenula totally immunonegative

(Fig. 3A). The habenula rested on a single layer of flat PCNA-ir

cells which made a continuum with PCNA-labeled cells caudally

in the dorsal thalamus and rostrally/rostroventrally in the

presumptive lamina terminalis area. In the ventral half of the

diencephalon, all around the preoptic recess, the presence of a

large number of labeled cells indicated high proliferative activity

(Fig. 3C). Similarly, the proliferative activity was particularly

high around the infundibular recess and there were numerous

PCNA-ir cells in the infundibular stalk (Fig. 3B). Labeled nuclei

were located in the ventricular and subventricular zones. The

optic tectum had now become a laminated structure in its rostral

portion and only the periventricular grey (ventricular and

subventricular zones) contained several layers of darkly stained

PCNA-ir cells (Fig. 3D). In its caudal extension, however, the

F. Raucci et al. / Journal of Chemical Neuroanatomy 32 (2006) 127–142 131

Fig. 3. Photomicrograph of a sagittal section (inset) of a premetamorphic tadpole with growing hindlimbs (Group III). Enlargements of boxed areas in the inset,

showing the distribution of PCNA-ir nuclei (arrows) in the dorsal thalamus (A), infundibular hypothalamus (B), preoptic area (C), rostral part of the optic tectum (D),

caudal part of the optic tectum (E) and rhombencephalon (F). Note the total absence of proliferating cells within the habenula (A) and the presence of labeled nuclei

exclusively in the periventricular grey layer of the rostrally stratified optic tectum (D) whereas the caudal part of the optic tectum is still a compact mass of labeled

nuclei (E). Abbreviations as in Fig. 5. Scale bars: (inset) 500 mm; (A and B) 100 mm; (C–F) 50 mm.

optic tectum was similar to that in the earlier stage of

development (no stratification or lamination had occurred)

and was entirely composed of a pseudostratified mass of ir cells

(Fig. 3E cf. Fig. 2D). The PCNA-labeled cells in the cerebellum

were numerous, a situation quite similar to that observed in the

earlier group of tadpoles. In the rhombencephalon, the PCNA-ir

periventricular lining, closely apposed to the posterior choroid

plexus (see Fig. 3, inset), was several cell-thick and numerous

scattered ir cells were observed at varying distances from it as if

they had migrated radially or tangentially (Fig. 3F).

F. Raucci et al. / Journal of Chemical Neuroanatomy 32 (2006) 127–142132

Fig. 4. Photomicrographs of a parasagittal (upper inset) and a midsagittal (lower inset) sections of an early prometamorphic tadpole (Group IV). Enlargements of the

boxed areas in the two insets illustrating the distribution of PCNA-labeled nuclei (arrows) in the optic tectum (A), telencephalon (B), infundibular hypothalamus (C)

and diencephalon (D). CP, pallial commissure; OC, optic chiasma; TL, lamina terminalis (terminal lamina); other abbreviations as in Fig. 5. Scale bars: (inset) 1 mm;

(A–D) 100 mm.

3.1.4. Group IV (stage 40)

In tadpoles with grown hindlimbs, the telencephalon

population of the PCNA-ir cells constituted a continuum, as

a pseudostratified periventricular lining (ventricular zone),

throughout the rostrocaudal and the dorsoventral axis of the

lateral ventricles (Fig. 4B). The optic tectum was very similar to

that observed in the tadpoles of Group III (Fig. 4A). Within the

thalamus, the majority of cells lining the ventricle dorsoven-

trally, were PCNA-ir. The lamina terminalis, now easily

distinguished, contained numerous PCNA-ir cells (Fig. 4B and

D). The proliferative activity around the preoptic recess was

very conspicuous as evidenced by the presence of a large

number of darkly stained PCNA-ir nuclei not only in the

ventricular and subventricular zones laterally and on the bottom

of the recess, but also in the magnocellular preoptic nucleus and

the suprachiasmatic nucleus (Fig. 4D). In sagittal sections, the

upward continuation of the PCNA-ir cells in the rostrolateral

ventricular zone of the preoptic recess appeared to be

interrupted by the pallial commissure; PCNA-ir cells were

again encountered along the bed nucleus of the pallial

commissure and the lamina terminalis (Fig. 4D). The posterior

tubercle is another region where there was abundance of

PCNA-ir cells denoting a high proliferative activity (Fig. 4D).

The infundibulum was comparatively more voluminous than

in the earlier stage of development and the cell layers

(ventricular and subventricular zones) lining the infundibular

recess was all PCNA-ir (Fig. 4C). The torus semicircularis area

was now easily distinguishable and its main body contained

numerous PCNA-ir cells; many ir cells surrounded it from

all directions.

3.1.5. Group V (stage 42)

In tadpoles with the forelimbs emerged, the distribution

profile of PCNA-ir nuclei within the brain was much modified

as compared to that observed in the earlier developmental

stages examined. The series of cross-sections of a brain in this

stage of development (Fig. 5) clearly showed that the

proliferating cells are located mainly along the ventricular

lining of the brain ventricles through the entire rostrocaudal

axis. It is also evident from this figure that there are regional

differences in the relative frequency of labeled cells along the

rostrocaudal or the dorsoventral axis in any given brain region.

Within the rostral telencephalon, the PCNA-ir cells forming the

pseudostratified lining of the ventral half of the lateral

ventricles appeared to be comparatively more abundant than

in the dorsal inner and lateral ventricular lining (Figs. 5A and

F. Raucci et al. / Journal of Chemical Neuroanatomy 32 (2006) 127–142 133

Fig. 5. Photomicrographs of cross-sections of the brain of a prometamorphic tadpole with forelimb emergence (Group V) at the levels indicated in the schematic

lateral view of the brain (inset). Arrows indicate the distribution of PCNA-ir nuclei. The left half of each brain section bears the labeling. ACP, anterior choroid plexus;

AMY, amygdala; AQ, aqueduct; CB, cerebellum; CG, central grey; E, epiphysis; EN, entopeduncular nucleus; HB, habenula; HR, hindbrain reticular formation; IN,

interpeduncular nucleus; INF, infundibulum; IR, infundibular recess; LEM, lemniscus; LV, lateral ventricle; MOT, medial optic tract; NA, nucleus accumbens; NI,

nucleus isthmi; ON, optic nerve; OT, optic tectum; OV, optic ventricle; PC, posterior commissure; PD, dorsal pallium; PL, lateral pallium; PM, medial pallium; POA,

preoptic area; PON, preoptic nucleus; PONm, magnocellular preoptic nucleus; POR, preoptic recess; PT, posterior tubercle; PTHN, posterior thalamic nucleus;

RHOM, rhombencephalon; SCN, suprachiasmatic nucleus; SCO, subcommissural organ; SGC, stratum griseum centrale; SGP, stratum griseum periventriculare; SL,

lateral septum; SM, medial septum; ST, striatum; TEGd, dorsal tegmentum; TEGr, rostral tegmentum; TEGv, ventral tegmentum; TEL, telencephalon; THd, dorsal

thalamus; THv, ventral thalamus; TS, torus semicircularis; TSL, laminar nucleus of the torus semicircularis; III, third ventricle; IV, fourth ventricle. Scale bar:

500 mm.

6A). At the level of the mid telencephalon, a pseudostratified

PCNA-ir ventricular lining was constantly observed particu-

larly along the bottom and the inner wall of the lateral

ventricles. Further caudally, the lateral ventricles begin to

narrow and continue more dorsolaterally in the posterior

extensions of the telencephalon; in this area there was a

conspicuous mass of PCNA-ir cells adjacent to the lateral

septum and medial septum and bulging into the lateral ventricle

(Fig. 5B). Dorsally, the lining of the lateral ventricles contained

comparatively fewer labeled cells than in an earlier stage of

F. Raucci et al. / Journal of Chemical Neuroanatomy 32 (2006) 127–142134

Fig. 6. Photomicrographs of sagittal sections of late prometamorphic tadpoles with forelimbs grown (Group V). (B) The control reaction with no labeled nuclei. (C)

The enlargement of boxed area in (A), showing that labeled nuclei (arrows) are concentrated in the ependymal lining of the most caudal medial part of the lateral

ventricle (LV) and the dorsal part of the third ventricle (III). Note that there are no ir nuclei in the habenula (HB). (D) A parasagittal section from another brain

showing in greater details the distribution of PCNA-ir nuclei around the third ventricle, in the anterior thalamic nucleus (A, arrow), lamina terminalis (TL) and the

dorsocaudal extremity of the cerebral hemisphere (thick arrow). No labeled nuclei in the habenula. (E) From a midsagittal section of the same brain as in (D), showing

the great concentration of proliferating cells in the caudalmost optic tectum (OT), torus semicircularis (TS), tegmentum (TEG) and inner molecular (ML) and outer

granular layer (GL) of the cerebellum. (F) Midsagittal section as in (E), showing the labeled nuclei (arrows) below and around the preoptic recess in the

suprachiasmatic (SCN) and magnocellular preoptic (PONm) nuclei; the epiphysis (E) is totally negative. CP, pallial commessure; TEG, tegmentum; other

abbreviations as in Fig. 5. Scale bars: (inset) 1 mm; (C–H) 100 mm.

development; here the labeled cells did not form a continuous

lining of the ventricles. Exception to this distribution pattern

was the large number of PCNA-ir cells located at the dorsal

rostralmost extremity of the lateral ventricles (see Fig. 6A). The

ventricular lining of the third ventricle contained a large

number of PCNA-ir cells. Seen dorsoventrally, the major

portion of the cell lining of this ventricle was PCNA-ir; cells

were unlabeled in the vicinity of the habenula (Fig. 5C and D),

at a level between ventral thalamus and suprachiasmatic

nucleus (Fig. 5D), and along the midlateral of the rostral

tegmental area (Fig. 5E). No PCNA-ir cells were observed

within the habenulae (Figs. 5C and D and 6C and D); evidently

their growth is supported by the supply of new cells originating

in the anterior and anterodorsal thalamic nuclei, areas rich in

PCNA-ir cells. The preoptic recess was lined with PCNA-ir

cells in its basal, basolateral and middle portions with the ir

cells continuing into the magnocellular nucleus rostrally and

the suprachiasmatic nucleus caudally (Figs. 5B–D and 6F).

These nuclei contained comparatively less PCNA-ir cells than

in Group IV tadpoles (see Fig. 4D). At the level of the

F. Raucci et al. / Journal of Chemical Neuroanatomy 32 (2006) 127–142 135

subcommissural organ, which itself was totally devoid of any

proliferating cells, the ventricular lining at the height of the

posterior thalamic nucleus was rich in proliferating cells

(Fig. 5E). As compared to earlier stages, the ventricular lining

of the infundibular recess (mainly along the ventral hypotha-

lamus) was richly laid with PCNA-ir cells (Fig. 5E). The

periventricular cell lining of the optic ventricles showed further

progress in its process of stratification in a rostrocaudal

direction; while there were still some PCNA-ir cells in the

periventricular lining in the anterior medial and mediolateral

portions, a conspicuous mass of PCNA-ir cells was observed in

the posterior part of the optic lobes continuing downward into

the torus semicircularis (Fig. 6E). Indeed, in the caudal part of

the mesencephalon, while the lateral extremity of the optic

ventricle was lined by numerous ir cells, the big mass of PCNA-

ir cells was located medially, lining the roof and the floor (Figs.

5F and 6E). The ventromedial mass of labeled cells made a

continuum with the outer granular layer of the cerebellum

(Figs. 5F–H and 6E). In cross-sections, it was evident that the

majority of the ir cells are concentrated near the midline

(Fig. 5F). In the cerebellum, whereas the PCNA-ir molecular

layer was pseudostratified medially, more laterally the labeled

cells constituted a single layer (Figs. 5G and H and 6E). The

Fig. 7. Photomicrographs from a sagittal section of a brain from metamorphosing ta

head) of the lateral ventricle showing few ir nuclei (arrows) in the ependyma. (B) L

nuclei and the inner nuclei rather ovoidal. (C) Labeled nuclei are concentrated in th

labeled nuclei in the granular layer (GL) and pseudostratified molecular layer (ML) w

rare. Other abbreviations as in Fig. 5. Scale bars: (A, B and D) 100 mm; (C) 100

periventricular lining of the aqueduct also appeared as a

pluristratified mass of PCNA-ir cells, except in its midventral

portion (Figs. 5F and 6E). In the rhombencephalon, the lining of

the fourth ventricle still contained many ir cells (Fig. 5G and

H). In contrast with all other brain areas, however, here the

proliferating cells were mostly located laterally rather than

medially (Fig. 5G and H).

3.1.6. Group VI (stages 44–45)

During the metamorphic climax, the different brain regions

had further differentiated. The PCNA-ir cells appeared

comparatively less numerous in several parts of the brain as

compared with the relative frequency of such cells in the brain

of earlier stages of development. In the telencephalon, the cell

lining of the basal part of the lateral ventricles was still

composed of many ir cells. Dorsally, in the most rostral portion,

however, the proliferative activity had decreased to a great

extent as compared with some early stages of development

(Fig. 7A). The immunostained nuclei in this part of the brain

appeared ovoidal or elongated and many of them appeared

displaced in a radial fashion. The PCNA-ir cells do not form a

continuous lining of the lateral ventricles; the labeled nuclei

rather formed a sort of patchwork, i.e., they were intermingled

dpole (Group VI). Rostral to the left. (A) Rostralmost, dorsal extension (arrow

amina terminalis (TL) with the periventricular layer made of labeled columnar

e innermost periventricular grey layer (arrows). (D) A single layer of columnar

ith ovoidal ir nuclei. Labeled nuclei in the central Purkinje cell layer (PUL) are

mm.

F. Raucci et al. / Journal of Chemical Neuroanatomy 32 (2006) 127–142136

F. Raucci et al. / Journal of Chemical Neuroanatomy 32 (2006) 127–142 137

with immunonegative cells. The terminal lamina (lamina

terminalis) still contained a large number of PCNA-ir cells with

those in contact with the third ventricle being columnar in shape

whereas the more distant ones were rather ovoidal in shape

(Fig. 7B). The region of the neuroepithelium immediately

beneath the lamina terminalis was practically devoid of PCNA-

ir cells. In the optic tectum, the stratification of tectal cells had

advanced throughout the rostrocaudal axis and the PCNA-ir

cells were now restricted to the innermost layers and formed a

pseudostratified ventricular lining (Fig. 7C). The granular layer

of the cerebellum was now composed of a single layer of

PCNA-ir columnar cells (Fig. 7D). The inner, molecular layer

was composed of a pseudostratified palisade of ovoidal PCNA-

ir nuclei (Fig. 7D). Rare PCNA-ir cells were observed in the

central layers of the cerebellar plate. We never observed ir

nuclei belonging to Purkinje cell types.

3.1.7. Adult

In the telencephalon, the most rostrally located PCNA-ir

cells were present along the dorsal and dorsolateral extension

of the ventricular lining. Similarly, although not as numerous

as they were in premetamorphic tadpoles, ir cells were also

present in the most caudal, dorsolateral extension of the

lateral ventricles and were localized exclusively in the dorsal

ventricular lining (Fig. 8A); such cells were typically

columnar and distributed either singly or in very small groups,

of two to three cells, and there were strongly stained as well

weakly stained nuclei (Fig. 8A). In the ventromedial and

ventrocaudal extensions of the lateral ventricles the ventricular

lining was rich in ir cells; whereas the bottom lining of the

dorsoventrally flattened ventricles contained several ir cells

interspersed with immunonegative cells, the roof lining was

composed of a continuum (rather elongated cells arranged in a

pseudostratified layer) of PCNA-ir cells (Fig. 8E). Exception-

ally, the outer lining of the caudalmost part of the cerebral

hemispheres, in close contact with the third ventricle, just

above the thalamic eminence, contained a few ir cells with a

rather flattened appearance (Fig. 8C). A little ventrally, several

small and rounded PCNA-ir cells were also observed in the

posterior wall of the medial septum in the vicinity of the

interventricular foramen (Fig. 8C). Slightly elongated ir cells

were also present near the bed nucleus of the pallial

commissure (Fig. 9E). Of the entire brain, by far the most

conspicuous concentration of PCNA-ir cells was observed in

the diencephalon. Starting rostrally in the preoptic area,

besides many haphazardly scattered (laterally/rostrally/dor-

sally) ir cells, the entire magnocellular preoptic nucleus area

contained numerous ir cells (Fig. 9F). Laterally and below the

preoptic recess there were many labeled nuclei and more

caudally a rather small group of labeled cells was present in the

Fig. 8. Photomicrographs of two parasagittal sections from an adult male brain (in

(arrows) in several brain areas: (A) scattered, weakly (black triangle) or strongly (ar

weakly (black triangle) or strongly stained (arrow) PCNA nuclei in the ventral linin

thalamic nucleus (VM) just behind the thalamic eminence (TE), some ir nuclei at the

caudalmost boundary of the cerebral hemisphere (arrow head); (D) numerous ir nuc

dorsal ependyma of the ventrolateral extension of the lateral ventricle (upper arrow)

nuclei in the infundibular ependyma lining the infundibular recess (arrows). Other

suprachiasmatic nucleus in its ventralmost portion (Fig. 9F).

The postchiasmatic infundibular hypothalamus was totally

devoid of any ir cell in the most rostral extension of the

ventricular lining of the infundibular recess. Only in its caudal

extension there were some labeled cells of elongated shape

(Fig. 8F, cf. Figs. 3B and 4C). In the diencephalon,

furthermore, the anterior thalamic nucleus contained a rather

conspicuous group of PCNA-ir nuclei of variable shapes

(Figs. 8D and 9D). The habenular nuclei were totally devoid

of ir nuclei and very few solitary labeled nuclei were observed

along the ventricular lining. In the thalamus, the ventromedial

nucleus is another site where a conspicuous group of PCNA-ir

cells was observed in its rostral part (Fig. 8C). Here again, in

its immediate rostral, caudal and dorsal vicinity displaced ir

cells were also observed. In the mesencephalon, no ir nuclei

were observed along the most rostral portion of the

periventricular grey layer of the optic ventricles, neither

medially nor laterally. Caudally, however, most of the labeled

nuclei in the ventral portion of this lining were concentrated

medially, whereas only a few labeled cells were seen in the

dorsal portion (Fig. 9C) and in the lateral extensions of the

optic ventricles (Fig. 8B). There were a few solitary scattered

labeled cells in the laminar nucleus whereas no PCNA-ir cells

were observed in the main body of the torus semicircularis.

Similarly, the periventricular area of the dorsal tegmentum

contained very few labeled cells. The isthmic nuclei were

devoid of any proliferating cell. In the cerebellum, the granular

layer contained many darkly stained PCNA-ir cells (Fig. 9B);

the labeled cells were located medially while the lateral

extensions of the cerebellar plate were totally devoid of such

cells. Some ir cells, stained rather weakly, were scattered in the

medial cerebellar area. It is noteworthy that the molecular layer

totally lacks any proliferative activity. This characteristic has

been verified in all adult brains, males and females. The

proliferative activity along the lining of the fourth ventricle, in

the rhombencephalon, was drastically low in the sense that

none or only very few labeled cells were observed in any single

sagittal section.

3.2. Western blot analysis

Western blot analyses were performed on crude extracts of

the frog brain (developmental stages and adult males and

females). The anti-PCNA antibody detected a positive 36 kDa

band in all samples. The bands detected in the adult brain of

males and females were weaker and a comparatively lower

optical density was recorded in these bands (Fig. 10). Groups

corresponding to premetamorphic (Groups III–V poll and

metamorphic stages (1 and 2) showed significantly higher

values (P < 0.01).

sets). Enlargements of boxed areas in the two insets showing PCNA-ir nuclei

rows) stained PCNA nuclei in the caudalmost dorsal lateral ventricle; (B) rare,

g of the caudolateral optic ventricle; (C) strongly ir nuclei in the ventromedial

caudal extremity of the medial septum (SM) and a few small nuclei at the dorsal,

lei in the anterior thalamic nucleus (A); (E) numerous columnar ir nuclei in the

with less labeled cells in the floor ependyma (lower arrow); (F) only few labeled

abbreviations as in Fig. 5. Scale bars: (inset) 1 mm; (A–F) 100 mm.

F. Raucci et al. / Journal of Chemical Neuroanatomy 32 (2006) 127–142138

Fig. 9. Photomicrograph of a midsagittal section of the same brain as in Fig. 8. Boxed areas in (A) are enlarged in (B–F). (B) Labeled nuclei in the medial part of the

granular layer of the cerebellum (arrows); empty triangles indicate some weakly stained nuclei at some distance from the granular layer. (C) Caudal optic tectum with

ir nuclei (arrows) in the basal ependyma of the optic ventricle; weakly stained nuclei (black triangle) are sometimes observed. (D) Darkly stained ovoidal nuclei

(arrows) in the anterior thalamic nucleus and some displaced nuclei as well. (E) ir nuclei near the bed nucleus of the pallial commissure (BNCP) and a few at the

caudal boundary of the medial septum (uppermost arrow). (F) ir nuclei (arrows) are abundant in the magnocellular preoptic nucleus; there are ir nuclei just below the

preoptic recess (POR), and a small group of labeled nuclei in the posterior wall of the recess. LAM, nucleus laminaris of torus semicircularis; other abbreviations as in

Fig. 5. Scale bars: (inset) 1 mm; (A–F) 100 mm.

4. Discussion

For the past 30 years and more, the neurogenic and

regenerative capacities of the amphibian nervous system have

been investigated repeatedly involving frogs, newts and

salamanders (see Bernocchi et al., 1990; Margotta et al.,

2000; Wullimann et al., 2005). Nevertheless, the present study,

using a widely accepted and valid proliferating cell marker

F. Raucci et al. / Journal of Chemical Neuroanatomy 32 (2006) 127–142 139

Fig. 10. Western blot analyses of PCNA protein in brain extracts of tadpoles

and adults of R. esculenta. Protein (30 mg/lane) was resolved by SDS-PAGE,

transferred to nitrocellulose membrane and incubated with antibody raised

against PCNA protein. A specific band of about 36 kDa was observed by

comparison with co-migrating size markers (Bio-Rad). The amount of PCNA

was quantified by densiometry using the Scan program and normalized by total

PCNA protein. The values shown represent the mean of three separate deter-

minations. Significant values: *P < 0.01. (1) Premetamorphic developmental

stages (Groups III–V); (2) metamorphic climax (Group VI); (3) adult males; (4)

adult females.

technique, is to our knowledge the first detailed analysis of the

neuroanatomical picture of the putative proliferating areas

within the larval and adult brain of an amphibian.

That the developing and adult green frog (R. esculenta) brain

produces a PCNA protein was confirmed by its easy detection

in Western blot analysis. Although we have not used any

technique to characterize the neural or glial cell phenotype it is

implied that in our study cells stained with PCNA express

markers selective for neurons or glia. This aspect will not be

discussed here; suffice it to mention that past reports have

suggested that proliferating progenitor cells in the vertebrate

brain express neural- or glial-specific phenotypes (Alvarez-

Buylla et al., 2002; Gould and Gross, 2002; Chapman et al.,

2006).

Cell proliferation in the brain of R. esculenta continues

throughout larval development, during metamorphosis and in

the adult. In the early and late tadpoles (prior to metamor-

phosis) of R. esculenta, several areas of the brain along the

rostrocaudal course show abundance of PCNA-ir cells implying

a high proliferative potential; these areas include the caudal part

of the medial septum contiguous with the caudalmost dorsal

pallium, anterior preoptic area (magnocellular preoptic nucleus

and the suprachiasmatic nucleus in particular), posterior

tuberculum, dorsomedial caudal optic tectum contiguous with

the torus semicircularis and the tegmentum. While a long-

itudinal organization of PCNA-ir cells in telencephalon is

apparently seen, we are not in a position to unequivocally

demonstrate that this distribution pattern could fit the paradigm

of a neuromeric organization of the forebrain. The meta-

morphic climax is the period during which there occurs an

apparent reduction in the proliferative potential of the brain.

Indeed, in the adult brain a reduction in the proliferative

potential of these areas becomes evident because of a great

reduction in the relative frequency of the PCNA-labeled cells;

an exception is the anterior preoptic area where a conspicuous

mass of labeled cells was observed, in both sexes. No sexual

dimorphism as to the relative frequency or distribution of the

PCNA-labeled cells in the adult was observed by us. We have

not made a seasonal analysis of the proliferative activity in R.

esculenta brain; however, Minelli et al. (1982) had earlier

suggested that there may be seasonal fluctuations in the

magnitude of such activity.

Although a careful analysis of serial sections of R. esculenta

brain leads us to consider that the brain cells originating in the

ventricular cell lining migrate tangentially as well as radially in

all brain areas, we have no data to distinguish between cells

being born in situ rather than migrating from the ventricular

lining. Pulse-chase techniques may be required to determine

which cells may be migrating. The proliferating cells do not

always form a continuous lining around the brain ventricles;

often they are found as single or small groups of cells scattered

among the unlabeled cells. Although PCNA-ir cells were

observed throughout much of the brain during development, in

the advanced larval stages (stages 42–45) the labeled cells were

mainly confined to the ventricular/subventricular areas. The

only other amphibian species as yet investigated in this regard is

the African clawed frog, X. laevis. During the early larval life of

this frog the periventricular cell layers were described to be

composed of most cells labeled with PCNA and whose relative

abundance decreased drastically during the advanced stages of

development (Wullimann et al., 2005). Almost a decade ago,

Filoni et al. (1995) had described the presence of numerous

cycling cells around the lateral ventricles in the brain of X.

laevis larvae and observed that they became drastically low in

number in the adult brain. Unlike X. laevis, however, in R.

esculenta the PCNA-ir nuclei in the brain of tadpoles

immediately prior to and during metamorphosis as well as

of adults stages were rather abundant, although comparatively

less than in the early tadpole stages.

It is surprising that in the larval and adult R. esculenta the

nucleus isthmi was never found to contain in its neuroanato-

mical perimeter any PCNA-ir cell, a situation in contrast with

that of X. laevis (Tay and Straznicky, 1980) and Lymnodynastes

dorsalis (Dann and Beazley, 1989) in which proliferating cells

were described in the nucleus isthmi; in the nucleus isthmi of

both species active neurogenesis was described until the

midlarval life. No mention of this nucleus was made in the

paper by Wullimann et al. (2005), however. Similarities in the

occurrence of proliferating cells in the developing and/or adult

amphibian brain, as recognized by one or the other technique

(e.g., 3H-thymidine, BrdU, PCNA), have been described for the

telencephlon, preoptic recess, hypothalamus, optic tectum and

F. Raucci et al. / Journal of Chemical Neuroanatomy 32 (2006) 127–142140

cerebellum (Filoni and Margotta, 1971; Gona, 1976; Dann and

Beazley, 1988; Chetverukhin and Polenov, 1993; Margotta

et al., 2000). That cell proliferation undergoes changes in the

central nervous system was also recently confirmed in the

auditory medulla of R. catesbeiana (Chapman et al., 2006).

These authors used BrdU technique and observed that the

number of proliferating cells significantly increases during

metamorphic climax but decreases immediately thereafter in

the postmetamorphic froglets. It was also ascertained in this

study that newly generated cells in the auditory medulla express

either glial- or neural-specific phenotypes.

Our description of the distribution pattern of the proliferative

activity in the adult brain is, in general terms, consistent with

that of Bernocchi et al. (1990); in fact, using 3H-thymidine to

label dividing cells in the R. esculenta brain, these authors

described high proliferative activity in the ependymal

(ventricular) layers of the lateral ventricles, and preoptic and

infundibular recesses. Since we used PCNA immunohisto-

chemistry and Bernocchi et al. (1990) used 3H-thymidine

labeling, we do not have stringent criteria to make a detailed

comparison. These authors furnished a generalized picture of

the proliferating areas in the adult brain and provided very few

details and described that during winter the proliferative

activity is extremely low (cf. Minelli et al., 1982). It is obvious

that research needs to be done in order to recognize and

characterize the factors that progressively downregulate the

proliferative activity in the developing amphibian brain, and

those which upregulate the proliferative activity when it may be

required for regeneration and/or normal adult neurogenesis/

gliogenesis as has been ascertained in various vertebrate

groups.

A comparison with other vertebrate groups begins with the

cyclostomes. In this group of the anamniote vertebrates, cell

proliferation domains (ventricular zones) were observed in

different neuroanatomical regions of the forebrain, midbrain

and hindbrain during embryonic and larval development, with

the proliferative activity becoming extremely scarce or totally

absent in most parts of the brain in the adult (Vidal Pizarro et al.,

2004; Villar-Cheda et al., 2005a,b). The proliferative activity

continues in the spinal cord, however. Unlike the green frog (R.

esculenta), in the sea lamprey (Petromyzon marinus), the

habenula and the pineal complex showed PCNA-ir proliferating

cells (Villar-Cheda et al., 2002). Among teleost fishes, in the

Senegal sole, the PCNA-ir proliferation zones (periventricular

cell linings) in the forebrain of the premetamorphic animals

were similar to those observed by us in R. esculenta: pallium

and subpallium, preoptic region, pretectum, dorsal and ventral

thalamus and hypothalamus (Pinuela et al., 2004). During and

after metamorphosis, however, the frequency of PCNA nuclei

decreased. In some other teleosts, the proliferation zones of the

developing brain corresponded to the same areas as in the sole

(see Wullimann and Puelles, 1999; Ekstrom et al., 2001;

Wullimann and Mueller, 2004). In contrast with R. esculenta, in

some teleostean fish species, however, the molecular layer of

the cerebellum was described to contain proliferating cells

(Zupanc et al., 1996; Ekstrom et al., 2001). In the gymnotiform

teleost, Apteronotus leptorhynchus, Zupanc et al. (1996)

described that labeled cells in the molecular layer of the

cerebellum ‘‘migrate’’ to the granular layer. Whether a similar

phenomenon, including the presence of labeled cells in the

cerebellar molecular layer, occurs in adult R. esculenta during a

period of the year other than June is not known to us. Reptiles

have not been ignored in this context. In fact, not only has

neurogenesis been described in some species during develop-

ment, its occurrence in adulthood has been reported in the

major subdivisions of the lizard forebrain, including the

olfactory bulbs, and in one species even in the cerebellum (see

Perez-Canellas and Garcıa-Verdugo, 1996; Garcia-Verdugo

et al., 2002; Pritz, 2003; Romero-Aleman et al., 2004). Cell

proliferation in birds has been described in the brain during

development and adult neurogenesis has been emphasized in

several species of birds, restricted principally to the tele-

ncephalon (see Garcia-Verdugo et al., 2002; Margotta et al.,

2005; Dewulf and Bottjer, 2005). In the canary, for example,

cell proliferation in the adult brain involves most of the major

divisions of the telencephalon (a seasonally oriented neurogen-

esis occurring in the song nuclei) while it was totally absent in

the hypothalamus, septum, thalamus, cerebellum, optic tectum

and brain stem (see Nottebohm, 1985; Alvarez-Buylla, 1990).

Generation of new cells in the brain has been described

throughout embryonic and prenatal development of the

mammalian nervous system where it is dramatically reduced

after birth; in the adult brain it has been described to occur in the

subventricular zone of the lateral ventricle and of hippocampus

(see Lois and Alvarez-Buylla, 1993; Ino and Chiba, 2000;

Rodrıguez-Perez et al., 2003; Garcia-Verdugo et al., 2002;

Bedard and Parent, 2004; Rankin et al., 2004). While cell

proliferation occurs across a wide range of areas in the

developing brain, in the adult it appears to be restricted to the

subventricular (subependymal) zone which is defined to be the

major source of neural stem cells at least among the amniotes.

Thus, amphibians and teleost fishes are probably the only

vertebrates known today in which all major subdivisions of the

brain remain involved in generation of new cells in the

adulthood. In these two vertebrate groups, furthermore, the

ventricular and subventricular zones are apparently involved in

the generation of new cells. It is plausible to suggest that during

development of the frog brain, the vast majority of its

constituent cells arise from neuronal and non-neuronal

precursor cells located in the ventricular/subiventricular

(ependymal/subependymal) lining of the brain ventricles all

along the rostrocaudal and the dorsoventral axis. The normal

adult brain, male or female, as a whole, is composed of

quiescent tissue as well as proliferating cells located mainly in

the periventricular cell linings leading us to infer that there is

potential for neurogenesis and/or gliogenesis in the adult frog.

This is consistent with the repeatedly demonstrated regen-

erative capacity of the amphibian brain in the past (see

Margotta et al., 2000, for earlier literature). The persistence of

proliferative activity in the brain during adulthood may lead us

to assume that cell proliferation may also be related to changes

in the frequency of certain peptidergic neuronal phenotypes,

such as FMRFamide or gonadotropin-releasing hormone

neurons in the forebrain, midbrain and hindbrain (see Rastogi

F. Raucci et al. / Journal of Chemical Neuroanatomy 32 (2006) 127–142 141

et al., 1990, 1998, 2001; Iela et al., 1994). These considerations

imply that newly differentiated neuropeptidergic neurons in

different parts of the adult brain may derive from committed

progenitors located within the closely lying proliferation zones.

In conclusion, a detailed neuroanatomical map of the

proliferation zones of the brain during development and

adulthood may play a crucial role in future studies on whether

the presence of a prominent number of proliferating cells in the

adult brain is a sign of a sustained growth or simply a

replacement of neurons/glia lost by apoptosis.

Acknowledgements

This work was supported by the Second University of

Naples, the University of Naples Federico II and in part by

PRIN 2003 (to RKR).

References

Alvarez-Buylla, A., 1990. Mechanism of neurogenesis in adult avian brain.

Experientia 46, 948–955.

Alvarez-Buylla, A., Seri, B., Doetsch, F., 2002. Identification of neural stem

cells in the adult vertebrate brain. Brain Res. Bull. 57, 751–758.

Bedard, A., Parent, A., 2004. Evidence of newly generated neurons in the

human olfactory bulb. Dev. Brain Res. 151, 159–168.

Benraiss, A., Arsanto, J.P., Coulon, J., Thouveny, Y., 1999. Neurogenesis during

spinal cord regeneration in adult newts. Dev. Genes Evol. 209, 363–369.

Bernocchi, G., Scherini, E., Giacometti, S., Mares, V., 1990. Premitotic DNA

synthesis in the brain of the adult frog (Rana esculenta L.): an autoradio-

graphic 3H-thymidine study. Anat. Rec. 228, 461–470.

Burd, G.D., Sein, V., 1998. Influence of olfactory innervation on neurogenesis in

the developing olfactory bulb of the frog, Xenopus laevis. Ann. N. Y. Acad.

Sci. 855, 270–273.

Chapman, J.A., Weinstein, J.L., Simmons, A.M., 2006. Cell proliferation in the

Rana catesbeiana auditory medulla over metamorphic development. J.

Neurobiol. 66, 115–133.

Chetverukhin, V.K., Polenov, A.L., 1993. Ultrastructural radioautographic

analysis of neurogenesis in the hypothalamus of the adult frog, Rana

temporaria, with special reference to physiological regeneration of the

preoptic nucleus. I. Ventricular zone cell proliferation. Cell Tissue Res. 271,

341–350.

Chiasson, B.J., Tropepe, V., Morshead, C.M., van der Kooy, D., 1999. Adult

mammalian forebrain ependymal and subependymal cells demonstrate

proliferative potential, but only subependymal cells have neural stem cell

characteristics. J. Neurosci. 19, 4462–4471.

Dann, J.F., Beazley, L.D., 1988. Development of the optic tecta in the frog

Limnodynastes dorsalis. Dev. Brain Res. 44, 21–35.

Dann, J.F., Beazley, L.D., 1989. Neurogenesis and cell death in the isthmic

nuclei of the frog Limnodynastes dorsalis. J. Comp. Neurol. 283, 28–37.

Dawley, E.M., Fingerlin, A., Hwang, D., John, S.S., Stankiewicz, C.A., 2000.

Seasonal cell proliferation in the chemosensory epithelium and brain of red-

backed salamanders, Plethodon cinereus. Brain Behav. Evol. 56, 1–13.

Del Grande, P., Franceschini, V., Minelli, G., Ciani, F., 1984. Matrix area

activity in the regenerative optic tectum of Rana esculenta. Z. Mikrosk. -

Anat. Forsch. 98, 72–80.

Del Grande, P., Franceschini, V., Minelli, G., Ciani, F., 1990. Mitotic activity of

the telencephalic matrix areas following optic tectum or pallial cortex lesion

in newt. Z. Mikrosk. -Anat. Forsch. 104, 617–624.

Dewulf, V., Bottjer, S.W., 2005. Neurogenesis within the juvenile zebra finch

telencephalic ventricular zone: a map of proliferative activity. J. Comp.

Neurol. 481, 70–83.

Ekstrom, P., Carl-Magnus, J., Liss-Mari, O., 2001. Ventricular proliferation

zones in the brain of an adult teleost fish and their relation to neuromere and

migration (secondary matrix) zones. J. Comp. Neurol. 436, 92–110.

Eriksson, P.S., Perfilieva, E., Bjork-Eriksson, T., Alborn, A.M., Nordborg, C.,

Peterson, D.A., Gage, F.A., 1988. Neurogenesis in the adult human hippo-

campus. Nat. Med. 4, 1313–1317.

Filoni, S., Margotta, V., 1971. A study of the regeneration of the cerebellum of

Xenopus laevis (Daudin) in the larval stages and after metamorphosis. Arch.

Biol. (Liege) 8, 433–470.

Filoni, S., Bernardini, S., Cannata, S.M., 1995. Differences in the decrease in

regenerative capacity of various brain regions of Xenopus laevis are related to

differences in the undifferentiated cell populations. J. Brain Res. 36, 523–529.

Franceschini, V., Del Grande, P., Minelli, G., Ciani, F., 1992. 6-H3 thymidine

uptake in the central nervous system of newt following lesion and cold

shock. J. Hirnforsch. 33, 235–239.

Garcia-Verdugo, J.M., Ferron, S., Flames, N., Collado, L., Desfilis, E., Font, E.,

2002. The proliferative ventricular zone in adult vertebrates: a comparative

study using reptiles, birds, and mammals. Brain Res. Bull. 57, 765–775.

Gona, A.G., 1976. Autoradiographic studies of cerebellar histogenesis in the

bullfrog tadpole during metamorphosis: the external granular layer. J.

Comp. Neurol. 165, 77–87.

Gorlick, D.L., Kelley, D.B., 1987. Neurogenesis in the vocalization pathway of

Xenopus laevis. J. Comp. Neurol. 257, 614–627.

Gosner, K.L., 1960. A simplified table for staging anuran embryos and larvae

with notes on identification. Herpetology 16, 183–190.

Gould, E., Gross, C.G., 2002. Neurogenesis in adult mammals: some progress

and problems. J. Neurosci. 22, 619–623.

Gould, E., Reeves, A.J., Graziano, M.S.A., Gross, C.G., 1999. Neurogenesis in

the neocortex of adult primates. Science 286, 548–552.

Iela, L., D’Aniello, B., di Meglio, M., Rastogi, R.K., 1994. Influence of

gonadectomy and steroid hormone replacement therapy on the gonadotro-

pin-releasing hormone neuronal system in the anterior preoptic area of the

frog (Rana esculenta) brain. Gen. Comp. Endocrinol. 95, 422–431.

Ino, H., Chiba, T., 2000. Expression of proliferating cell nuclear antigen

(PCNA) in the adult and developing mouse nervous system. Mol. Brain

Res. 78, 163–174.

Jaskulski, D., deRiel, J.K., Mercer, W.E., Calabretta, B., Baserga, R., 1988.

Inhibition of cellular proliferation by antisense oligodeoxynucleotides to

PCNA cyclin. Science 240, 1544–1546.

Lois, C., Alvarez-Buylla, A., 1993. Proliferating subventricular zone cells in the

adult mammalian forebrain can differentiate into neurons and glia. Proc.

Natl. Acad. Sci. U.S.A. 90, 2074–2077.

Margotta, V., Morelli, A., 1996. Encephalic matrix areas and post-natal

neurogenesis under natural and experimental conditions. Anim. Biol. 5,

117–131.

Margotta, V., Morelli, A., Alfei, L., 1999a. PCNA positivity in the telencephalic

matrix areas in the adult of a lizard, Podarcis sicula. J. Brain Res. 3, 271–276.

Margotta, V., Morelli, A., Alfei, L., 1999b. PCNA positivity in the telencephalic

matrix areas in the adult of a newt, Triturus carnifex. J. Brain Res. 39, 523–

528.

Margotta, V., Morelli, A., Alfei, L., 2000. PCNA positivity in the telencephalic

matrix areas in the adult of a frog, Rana esculenta. Rend. Fis. Acc. Lincei

11, 185–196.

Margotta, V., Caronti, B., Morelli, A., Alfei, L., 2005. Signs of normal

proliferation in the telecephalon of adult male songbirds (Serinus serinus),

as shown by PCNA-positivity. Ital. J. Anat. Embryol. 110, 33–44.

Martinez, S., Puelles, L., 2000. Neurogenetic compartments of the mouse

diencephalon and some characteristic gene expression patterns. In: Goffinet,

A.M., Rakic, P. (Eds.), Mouse Brain Development. Springer, Berlin, pp.

91–106.

Minelli, G., Del Grande, P., Franceschini, P., 1982. Uptake of 6-H3 thymidine in

the normal and regenerating CNS of Rana esculenta. Z. Mikrosk. -Anat.

Forsch. 96, 201–213.

Nottebohm, F., 1985. Neuronal replacement in adulthood. Ann. N. Y. Acad. Sci.

457, 143–161.

Onishchenko, L.S., Tverskikh, N.M., Chetverukhin, V.K., 1983. Autoradio-

graphic study of the proliferative activity of the preoptic recess ependyma in

yearling and juvenile common frogs. Tsitologiia 25, 98–101.

Perez-Canellas, M.M., Garcıa-Verdugo, J.M., 1996. Adult neurogenesis in the

telencephalon of a lizard: a [3H] thymidine autoradio9graphic and bromo-

deoxyuridine immunocytochemical study. Dev. Brain Res. 93, 49–61.

F. Raucci et al. / Journal of Chemical Neuroanatomy 32 (2006) 127–142142

Pinuela, C., Rendon, C., Gonzales de Canales, M.L., Sarasquete, C., 2004.

Development of the Senegal sole, Solea senegalensis forebrain. Eur. J.

Histochem. 48, 377–384.

Polenov, A.L., Chetverukhin, V.K., 1993. Ultrastructural radioautographic

analysis of neurogenesis in the hypothalamus of the adult frog, Rana

temporaria, with special reference to physiological regeneration of the

preoptic nucleus. II. Types of neuronal cells produced. Cell Tissue Res. 271,

351–362.

Pritz, M.B., 2003. Cell proliferation during early hindbrain development in

Alligator. Brain Behav. Evol. 62, 193–200.

Ramirez, C., Nacher, J., Molowny, A., Sanchez-Sanchez, F., Irurzun, A., Lopez-

Garcia, C., 1997. Photoperiod-temperature and neuroblast proliferation-

migration in the adult lizard cortex. NeuroReport 8, 2337–2342.

Rankin, S.L., Partlow, G.D., McCurdy, R.D., Giles, E.D., Fisher, K.R., 2004.

The use of proliferating cell nuclear antigen immunohistochemistry

with a unique functional marker to detect postnatal neurogenesis in par-

affin-embedded sections of the mature pig brain. Brain Res. Protoc. 13,

69–75.

Rastogi, R.K., D’Aniello, B., Pinelli, C., Fiorentino, M., Di Fiore, M.M., di

Meglio, M., Iela, L., 2001. FMRFamide in the amphibian brain: a com-

prehensive survey. Microsc. Res. Tech. 54, 158–172.

Rastogi, R.K., di Meglio, M., Iela, L., 1990. Immunoreactive luteinizing

hormone-releasing hormone in the frog (Rana esculenta) brain: distribution

pattern in the adult, seasonal changes, castration effects, and developmental

aspects. Gen. Comp. Endocrinol. 78, 444–458.

Rastogi, R.K., Meyer, D.L., Pinelli, C., Fiorentino, M., D’Aniello, B., 1998.

Comparative analysis of GnRH neuronal systems in the amphibian brain.

Gen. Comp. Endocrinol. 112, 330–345.

Reznikov, K.I., Maliovanova, S.D., 1979. Cell proliferation and migration in the

roof of the mesencephalon (tectum) in Xenopus laevis. II. Cell proliferation

and differentiation of the tectum. Ontogenez 10, 350–358.

Richter, W., Kranz, D., 1981. Autoradiographische Untersuchungen der post-

natalen Proliferationsaktivitat in den Matrixzonen des Telencepahlons und

des Diencephalons neim Axolotl (Ambystoma mexicanum), unter Beruck-

sichtigung der Proliferation im ilfactorischen Organ. Z. Mikrosk. -Anat.

Forsch. 95, 883–904.

Rietze, R., Poulin, P., Weiss, S., 2000. Mitotically active cells that generate

neurons and astrocytes are present in multiple regions of the adult mouse

hippocampus. J. Comp. Neurol. 424, 397–408.

Rodrıguez-Perez, L.M., Perez-Martın, M., Jimenez, A.J., Fernandez-Llebrez,

P., 2003. Immunocytochemical characterisation of the wall of the bovine

lateral ventricle. Cell Tissue Res. 314, 325–335.

Romero-Aleman, M.M., Monzon-Mayer, M., Yanes, C., Lang, D., 2004. Radial

glial cells, proliferative periventricular cells, and microglia might contribute

structural repair in the cerebral cortex of the lizard Gallotia galloni. Exp.

Neurol. 188, 74–85.

Straznicky, K., Gaze, R.M., 1972. The development of the tectum in Xenopus

laevis: an autoradiographic study. J. Embryol. Exp. Morphol. 28, 87–115.

Tay, D., Straznicky, C., 1980. The development of the nucleus isthmi in

Xenopus: an autoradiographic study. Neurosci. Lett. 16, 313–318.

Tsurimoto, T., 1999. PCNA binding proteins. Front. Biosci. 4, 849–858.

Uray, N.J., Gona, A.G., Hauser, K.F., 1987. Autoradiographic studies of

cerebellar histogenesis in the premetamorphic bullfrog tadpole: I. Genera-

tion of the external granular layer. J. Comp. Neurol. 266, 234–246.

Uray, N.J., Gona, A.G., Hauser, K.F., 1988. Autoradiographic studies of

cerebellar histogenesis in the premetamorphic bullfrog tadpole: II. Forma-

tion of the interauricular granular band. J. Comp. Neurol. 269, 118–129.

Valero, J., Weruaga, E., Murias, A.R., Recio, J.S., Alonso, J.R., 2005. Pro-

liferation markers in the adult rodent brain: bromodeoxyuridine and pro-

liferating cell nuclear antigen. Brain Res. Protoc. 15, 127–134.

Vidal Pizarro, I., Swain, G.P., Selzer, M.E., 2004. Cell proliferation in the

lamprey central nervous system. J. Comp. Neurol. 469, 298–310.

Villar-Cheda, B., Perez-Costas, E., Melendez-Ferro, M., Abalo, X.M., Rodrı-

guez-Munoz, R., Anadon, R., Rodicio, M.C., 2002. Proliferating cell

nuclear antigen (PCNA) immunoreactivity and development of the pineal

complex and habenula of the sea lamprey. Brain Res. Bull. 57, 285–287.

Villar-Cheda, B., Abalo, X.M., Anadon, R., Rodicio, M.C., 2005a. The teg-

mental proliferation region in the sea lamprey. Brain Res. Bull. 66, 431–435.

Villar-Cheda, B., Perez-Costas, E., Melendez-Ferro, M., Abalo, X.M., Rodri-

guez-Munoz, R., Anadon, R., Rodicio, M.C., 2005b. Cell proliferation in the

forebrain and midbrain of the sea lamprey. J. Comp. Neurol. 494, 986–1006.

Wullimann, M.F., Knipp, S., 2000. Proliferation pattern changes in the zebrafish

brain from embryonic through early postembryonic stages. Anat. Embryol.

202, 385–400.

Wullimann, M.F., Mueller, T., 2004. Identification and morphogenesis of the

eminentia thalami in the zebrafish. J. Comp. Neurol. 471, 37–48.

Wullimann, M.F., Puelles, L., 1999. Postembryonic neural proliferation in the

zebrafish forebrain and its relationship to prosomeric domains. Acta

Embryol. 199, 329–348.

Wullimann, M.F., Rink, E., Vernier, P., Schlosser, G., 2005. Secondary neu-

rogenesis in the brain of the African clawed frog, Xenopus laevis, as

revealed by PCNA, delta-1, neurogenin-related-1, and NeuroD expression.

J. Comp. Neurol. 489, 387–402.

Yoshino, J., Tochinai, S., 2004. Successful reconstitution of the non-regenerat-

ing adult telencephalon by cell transplantation in Xenopus laevis. Dev.

Growth Differ. 46, 523–534.

Zupanc, G.K.H., 1999. Neurogenesis, cell death and regeneration in the adult

gymnotiform brain. J. Exp. Biol. 202, 1435–1446.

Zupanc, G.K.H., 2001. Adult neurogenesis and neuronal regeneration in the

central nervous system of teleost fish. Brain Behav. Evol. 58, 250–275.

Zupanc, G.K.H., Horschke, I., 1995. Proliferation zones in the brain of adult

gymnotiform fish: a quantitative mapping study. J. Comp. Neurol. 353, 213–

233.

Zupanc, G.K.H., Horschke, I., Ott, I., Rascher, G.S., 1996. Postemrbyonic

development of the cerebellum in gymnotiform fish. J. Comp. Neurol. 370,

443–464.