Premitotic DNA synthesis in the brain of the adult frog Rana esculenta L.An autoradiographic 3H-thymidine studyкод для вставкиСкачать
THE ANATOMICAL RECORD 228:461-470 (1990) Premitotic DNA Synthesis in the Brain of the Adult Frog (Rana esculenta L.): An Auto radiographic 3H-Thy midine Study GRAZIELLA BERNOCCHI, ELDA SCHERINI, SILVIA GIACOMETTI, AND VLADISLAV MARES Department of Animal Biology, University of Pavia, The Center for Histochemistry of Italian National Research Council, Pavia, Italy (G.B., E.S., S.G.); Institute of Physiology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia (V.M.) ABSTRACT Replicative synthesis of DNA in the brain of the adult frog was studied by light microscope autoradiography. Animals collected during the active period (MayJune) and in hibernation (January) were used. In active frogs, 3Hthymidine labelling occurred mainly in the ependymal cells which line the ventricles. The mean labelling index (LI%)was higher in the ependyma of the lateral and fourth ventricles than in the ependyma of the lateral diencephalon and tectal parts of the mesencephalon. In the recessus infundibularis and preopticus the number of labelled cells (LCs) was several times greater than in the lateral parts of the third ventricle. LCs were seen subependymally only occasionally. The incidence of LCs in the parenchyma of the brain was much lower in most regions than in the ventricular ependyma; LCs were mainly small and, from their nuclear morphology, they were glial cells. The LI% reached the highest value in the septum hippocampi and in the nucleus entopeduncularis. In these locations, LCs were larger and closer in size to the nerve cells of these regions. From comparison with data obtained earlier in the brain of mammals, it is evident that the distribution of proliferating cells in the olfactory and limbic system is phylogenetically conservative. The occurrence of pyknotic cells in the same areas which contain LCs, suggests that cell division reflects in part the process of cell renewal observed in mammals. However, proliferating cells could also be linked to the continuous growth observed in non-mammalian vertebrates. In hibernating frogs, LCs and pyknoses were not seen or were found occasionally, which further indicates the functional significance of both processes. Several studies have reported cell division in discrete brain regions of non-mammalian vertebrates (Kirsche, 1967; Kranz and Richter, 1970a, 1970b; Richter and Kranz, 1970a, 1970b, 1971; Polenov et al., 1972; Chetverukin, 1978; Raymond and Easter, 1983; LopezGarcia et al., 1988; Yanes Mendez et al., 1988). From these studies, it appears that cell proliferation in the brain of non-mammals differs substantially from that in mammals (Smart and Leblond, 1961; Altman, 1963, 1966,1969; Altman and Das, 1964; Dalton et al., 1968; Hommes and Leblond, 1969; Kaplan and Hinds, 1980; Korr, 1980; Mares, 1986). The main difference appears to be in the proliferation of ependymal cells, both under normal conditions and during healing andlor repair. In contrast to mammals, a part of the ependyma in the adult brain of most non-mammalian vertebrates retains its matrix character, which can be activated after damage (Kranz and Richter, 1971). The residual matrix is distributed unevenly along the brain ventricles and, as shown in Teleostei by 3H-thymidine autoradiography (Kranz and Richter, 1970a, 1970b; Richter and Kranz, 1970a, 1970b, 1971), it is expressed at a similar, though low, level for most of the animal's life. Proliferation of cells in the subependymal regions, 0 1990 WILEY-LISS, INC. which is considerable in some parts of the mammalian brain, is much lower (Kirsche, 1967; Fleischauer, 1972; Polenov et al., 1972). The residual cell proliferation, resulting in long-lasting formation of new nonneuronal and probably neuronal cells, leads to continuous growth of the brain and increase in the total cell number in Teleostei (Raymond and Easter, 1983). Much less is known about proliferation of more differentiated cells in the brain parenchyma of adult nonmammals. According to autoradiographic studies performed mainly on fishes, cell division occurs in a small number of parenchymal glial cells (Kranz and Richter, 1970a, 1970b; Richter and Kranz, 1970a, 1970b, 1971; Reznikov, 1981; Raymond and Easter, 1983) and perhaps in neurons of some brain regions, e.g., cerebellum and optic tectum (Rahmann, 1968; Raymond and Easter, 1983). Received October 17, 1989; accepted April 27, 1990. Address reprint requests to Prof. Dr. Graziella Bernocchi, Dipartimento di Biologia Animale, Laboratorio di Istologia, Piazza Botta 10, 27100 Pavia, Italy. 462 G. BERNOCCHI ET AL. TABLE 1. Distribution of mean labelling index (LI%)in different brain regions of adult frogs during active period Brain vesicles Telencephalon Diencephalon Mesencephalon Metencephalon Myelencephalon Regions Lateral ventricles (E) Bulbus olfactorius (P) Pallium mediale (P) Pallium dorsale and laterale (P) Septum hippocampi (P) I11 ventricle (E) Recessus preopticus (E) Recessus infundibularis (E) Nucleus entopeduncularis (P) Tectum ventricle (E) Aqueductus Silvii (E) Tectum opticum (P) IV ventricle (E) Cerebellum (P) Medulla oblongata (P) LI% (2s.d.) 1.33 2 0.45 0.65 i 0.56 0.15 f 0.25 <0.01 3.72 f 0.54 0.17 2 0.15 0.92 & 0.31 1.86 2 0.09 15.32 3.18 0.20 2 0.28 0.18 t 0.25 <0.01 1.47 2 0.50 0.19 & 0.32 0.86 f 0.35 * Abbreviations: E, ependymal layer; P, parenchyma. The data available so far do not provide a complete Processing and Evaluation of Labelled and Pyknotic Cells picture of the rate of cell proliferation within the whole After killing, the brains were removed immediately brain, especially of amphibians. A more complete de- and fixed in Carnoy solution (6:3:1) for 12 hours. Hisscription is necessary for comparison of this process in tological sections (4 pm thick) of paraffin-embedded phylogenetically distant species. This could help t o re- whole brains were cut in the sagittal and transverse veal evolutionary links in residual cell proliferation planes. The slides were covered with Kodak ARlO and its possible functional significance in the brain of Stripping Film and exposed for 10-14 days. With this adults. Similarly, the morphology of cells engaged in exposure time, well-labelled cells were obtained with a premitotic DNA synthesis needs further specification. reasonably low background. Evaluation was performed Further, the occurrence of cell death, normally present on toluidine blue stained autoradiograms. Cells were in mammalian nervous system (Sturrock, 1979; Pan- considered as labelled when a t least 10 silver grains nese, 1981) has not yet been documented for non-mam- were present over their nuclei, though the majority of malian vertebrates. the LCs were heavily labelled (more than 40 silver This situation, together with our earlier pilot obser- grains). vations of DNA synthesis in a group of neuron-like Approximately one section out of each three to ten large cells in certain parts of the frog brain (Bernocchi (proportionally to the size of the brain region) was evalet al., 19851, have led us: 1)to study more systemati- uated per animal. The total numbers of LCs and unlacally in this species the cell proliferation in ventricle belled cells were counted by scanning a t a magnificalining and brain parenchyma by pulse 3H-thymidine tion of ~ 1 , 0 0 0around the ventricles and in the autoradiography; 2) to compare data from animals dur- parenchyma of individual brain regions separately. ing active period and winter inactivity; and 3) to inves- The labelling index (LI%) was calculated as the pertigate the relationship between the frequency of la- centage of LCs in respect to the total number of cells in belled cells (LCs) and that of pyknotic cells in the two the individual brain regions. The LI% values (Table 1) periods of the frog annual cycle. were calculated as mean values from the means (-t s.d.) measured in individual animals. The drawings of the MATERIALS AND METHODS distribution of LCs (Figs. 1,2) were obtained from four Animals superimposed sections of one representative active Experiments were carried out with adult frogs (Rana frog. The evaluation of the frequency of pyknotic cells esculenta L.) of about 7 cm length (legs excluded), col- was limited to the areas with the highest or the lowest lected in their natural environment in May and June LI% in active animals. The hematoxylin eosin stained (20-25"C), i.e., during the period of activity, and kept specimens were used to distinguish these cells from in the laboratory for 1 month before the experiment. erythrocytes. Other animals were collected in January (0-5"C), during underground hibernation, and used immediately. RESULTS The animals (six specimens for the active period and four for hibernation) were injected with 5 pCi/g body 3H-Thymidine Incorporation weight of 3H-thymidine (specific activity 865 GBq/ mM, UVVVR, Prague) in the inguinal lymphatic sac and killed by decapitation 2 hours later. After injection Active frogs The distribution of 3H-thymidine LCs in different both active and hibernating animals were kept a t room temperature (23°C). Animals without injections (three regions of the frog brain is schematically shown in Figspecimens for the active period and two for hiberna- ures 1, 2. The majority of the DNA-synthesizing cells tion) were used for morphological description and were heavily labelled. Their incidence and cytological characteristics will be given below. counting of pyknotic cells. CELL PROLIFERATION IN THE BRAIN OF ADULT FROGS Regional differences in DNA synthesis of ependymal cells. The brain ventricles of R. esculenta are lined by ependymal cells similar to those described by Paul (1967) for Rana temporaria. The main cell types are unipolar and bipolar tanycytes (gliocytes). Unipolar tanycytes show a central nucleus and cytoplasm reduced to a thin rim towards the ventricle lumen, while bipolar tanycytes have an eccentric nucleus. In some places cells of the wedge-shaped ependymal type (columnar tanycytes), i.e., the “Furchenependym” of Paul (1967), are found. Regressive modified ependymal cells (epithelial cells) with flat nuclei close to the ventricles are also present. In general, LCs were mainly ependymal. Subependymally, LCs were seen only occasionally. Because of the small number of labelled subependymal cells, quantitative evaluation of LCs was done only for proper ependymal cells. 463 Regional differences in DNA synthesis in the brain parenchyma. In most of the brain parenchyma LCs were rare (Table 1).They were numerous (LI% 3.72) in the nuclei of septum hippocampi (Fig. 5A,B), which are located dorsally, near the meningeal envelopes. These nuclei consist of cells which resemble neurons. They are of medium size with roundish nuclei, in which chromatin is packed in small clumps. Some of these cells were labelled quite heavily. In the pallium dorsale and laterale, there were few or no LCs. However, in the remaining pallium mediale (primordium hippocampi), LCs occurred more frequently (LI%0.15) and they were similar t o nerve cells in size. LCs were also scattered in the bulbus olfactorius (LI% 0.65). There was a cluster of LCs (LI% 15.32) near the preoptic area (Fig. 5C,D). In this location, which corresponds to the nucleus entopeduncularis, LCs were large, with a pale and roundish nucleus. 1. The telencephalic ventricles are lined mainly by a Inside the cerebellum, there were few LCs (LI% monostratified ependyma; dorsally and ventrally, 0.19). The cells were small and located mainly in deep pluristratified ependyma can be found. Ependymal parts of the internal granule layer. LCs were very rare cells of the lateral ventricles are mainly unipolar tany- in the mesencephalic tectum (<0.01%). cytes. Dorsally, there are bipolar tanycytes and, both LCs were also present in the parenchyma of the medorsally and ventrally, some wedge-shaped ependymal dulla oblongata (LI% 0.86) and they appeared to be cells are present. glia. LCs were mainly unipolar tanycytes. An average Brain accessories. In all locations, endothelial cells or LI% was 1.33 (Table 1).LCs were not evenly distrib- cells accompanying blood vessels were occasionally lauted. The highest frequency of LCs was found in the belled. LCs frequently occurred in the choroid plexi of ventral and medial parts of the ventricular wall (Fig. 3A,B), especially at the middle of the anterior-posterior axis of the telencephalon. The DNA-synthesizing cells Abbreviations occurred often in pairs or small clusters. 2. In the diencephalon, ependymal cells are mono- AMY amygdala aqueductus Sylvii stratified laterally and pluristratified in the recessus AS bulbus olfactorius preopticus. Laterally, the ependymal lining is formed BO cerebellum CE by unipolar tanycytes and, only dorsally, by regressive CH chiasma opticum modified ependymal cells. In some places subependy- GL glomerula olfactoria granular layer ma1 cells are present. The ependymal cells of the reces- GR habenula sus preopticus and recessus infundibularis are mainly HA infundibulum unipolar tanycytes; bipolar tanycytes and regressive INF lateral ventricle LV ependymal cells occur occasionally in these localities. MI layer of mitrial cells nucleus accumbens septi The mean LI% (Table 1) for the lateral parts of the NAS nucleus centralis thalami third ventricle was low (0.17) and the LCs were mainly NCT nucleus dorsalis septi of the unipolar tanycyte type; some labelled regressive NDS nucleus entopeduncularis NE ependymal cells were also found. In the recessus pre- NID nucleus infundibularis dorsalis nucleus interpeduncularis opticus and in the recessus infundibularis (Fig. 4A,B), NIS nucleus infundibularis ventralis LCs were mainly unipolar tanycytes. The LI% values NIV nucleus lateralis thalami NLT in the latter two regions (0.92 and 1.86) exceeded by NMS nucleus medialis septi several times the values for the lateral parts of the NPT nucleus posterior thalami nucleus princeps tori semicirculari third ventricle. In the recessus preopticus some la- NPTS nucleus ventromedialis thalami belled bipolar tanycytes were found. Occasionally LCs NVMT plexus choroideus PCH were also found subependymally near both recesses. pallium dorsale PD 3. In the mesencephalon, the tectal ventricle and the PG pretectal gray pallium laterale aqueductus Silvii are covered mostly by monostratified PL pallium mediale ependyma formed by unipolar tanycytes; in the dorsal PM preoptic recessus organ PRO part of the aqueduct, some wedge-shaped cells occur. paraventricularis organ PVO The LI% values (Table 1)were low (0.20 and 0.18) in RI recessus infundibularis substantia grisea centralis SGC both locations (Fig. 3C,D). septum hippocampi 4. In the metencephalic ventricle, the lateral SSTH striatum ependyma appears pluristratified. The cells are unipo- TEO tectum opticum lar tanycytes and regressive ependymal cells. Ven- TO tractus opticus ventricula tecti trally, some of the ependymal cells are wedge-shaped. VT nucleus nervi trigemini The LI% amounted to 1.47 (Table 1); LCs were V VI nucleus of the 6th nerve mainly of regressive type. In the ventral regions, mi- VII nucleus motorius nervi facialis toses were observed. VIII nucleus vestibularis 464 G. BERNOCCHI ET AL. A D G F H d7 M 0 N P I L K J E L Q R Fig. 1. Schematic drawings of transverse sections through frog brain taken at different levels in the rostra1 (A) to caudal (R) direction as illustrated by the drawing at the right lower side. The distribution of 3H-thymidine LCs is shown in the ventricular wall and the parenchyma. Each triangle indicates the presence of one LC; a black dot corresponds to three LCs. For each level, four sections from one representative animal were superimposed. A,B,C,D,E,F,G,H telencephalon; I: telencephalon-diencephalon;J,K: diencephalon; L,M: dien- cephalon-mesencephalon; N,O: mesencephalon; P,Q: metencephalonmielencephalon; R mielencephalon. LCs were highly frequent in C: the ventricle lining of the telencephalon, in F , G the recessus preopticus, and in M: the recessus infundibularis. Several LCs are present in E,F: the nuclei of septum hippocampi and in E, F, G the nucleus entopeduncularis. LCs frequently occur in I,O,Q,R the choroid plexi, and in E,F,J the leptomeninges. Scale bar = 1 mm. each ventricle (Fig. lI,Q,R), both in the stromal and the epithelial compartments. In the leptomeninges, LCs appeared mainly on the surface of the ventral part of the brain, especially in its more anterior parts (Fig. lE,J). Hibernating frogs Histologically, no gross differences from active animals were noticed, although the cellular regions adjacent to the recessus preopticus and infundibularis tended to be smaller. CELL PROLIFERATION IN THE BRAIN OF ADULT FROGS 465 1.3%, in the septum hippocampi 2.3%, in the nucleus entopeduncularis lo%, in the recessus preopticus 0.6%, and 1.5%in the lateral ventricles. During hibernation, the number of pyknotic cells decreased several fold in all regions. In the lateral ventricles, they were 0.1% and in the pallium mediale 0.5%. In the other regions, in which even higher values were found in active animals (septum hippocampi, nucleus entopeduncularis), there were practically no pyknotic cells. DISCUSSION Topological and Cytological Comments C L.23 D Fig. 2. Schematic drawings of sagittal sections through one half frog brain taken at different levels in A the lateral to D: the medial direction. The distribution of 3H-thymidine LCs is shown in the ventricular wall and the parenchyma. Each triangle indicates the presence of one LC; a black dot corresponds to three LCs. For each level, four sections from one representative animal were superimposed. Note the high frequency of LCs in the ventricle lining of A,B: the telencephalon and D in the nuclei of septum hippocampi. Scale bar = 1 mm. In comparison to active animals, LCs were much fewer in both ventricular and extraventricular regions; in the latter, no LCs were found in most sections and isolated LCs were observed only in the ependymal regions. Because of the very small number of the LCs, no quantitative comparison of labelling differences could be done. Pyknotic cells in active and hibernating animals. Cells with highly basophilic round or irregular nuclei with a thin rim of hypereosinophilic cytoplasm, typical of cells undergoing spontaneous death but different than erythrocytes, were seen. During the active period, the frequency of pyknotic cells in the pallium mediale was The location of 3H-thymidine LCs and sporadic mitoses indicates that proliferation of cells in adult frog brain takes place mainly within the ependymal lining. This is similar to adult fishes, other amphibians, and reptiles (Kirsche, 1967; Kranz and Richter, 1970a, 1970b;Richter and Kranz, 1970a, 1970b, 1971; Polenov et al., 1972; Chetverukhin, 1978; Lopez-Garcia et al., 1988; Raymond and Easter, 1983; Yanes Mendez et al., 1988), while in mammals the ependyma is mitotically less active. For instance, in the forebrain ventricles of the adolescent rat, the mean LI% is 0.34 (Chauhan and Lewis, 1979). As in frogs, the values for the third ventricle of rats were much lower (0.03%, Chauhan and Lewis, 1979). As shown in this study, the incidence of LCs also varies greatly along the ventricular border of the frog brain. The higher mean LI% values observed in the hypothalamic recessus preopticus and infundibularis and in the telencephalic ventricles are on the whole in agreement with earlier autoradiographic data obtained for some species of Teleostei (Kranz and Richter, 1970a, 1970b). In contrast to Teleostei, we found fewer LCs in the tectal and metencephalic ventricles of frogs. Cytological classification of LCs by light microscope autoradiography is limited. Nevertheless, it seems apparent that they are mostly tanycytes (gliocytes) of unipolar type. DNA synthesis in bipolar tanycytes has been observed in the hypothalamic preoptic recessus and in regressing types of ependymal cells in the diencephalic and metencephalic ventricles. Subependymally, LCs are much rarer. This is in accord with the very limited amount of subependymal matrix in non-mammals (Kirsche, 1967; Fleischauer 1972) and is in contrast to some parts of the mammalian brain. In mice and rats, where the residual subependyma1 layer is clearly visible near the lateral wall of the forebrain ventricles (Smart, 1961; Paterson et al., 1973), the average LI% is 15% to 20% (Smart and Leblond, 1961; MareS, 1975, 1986). The present study has shown that LCs also occur in the differentiated parenchyma of the adult frog brain. Their incidence in some regions, such as pallium mediale and medulla oblongata (0.15% and 0.86%, respectively) is comparable to that observed in mice (Mare6 and Lodin, 1974; Mares et al., 1975). The incidence of LCs in the cerebellum of frogs is close to the values observed in other regions (0.19%), while in mice it is several times lower (Mares, 1975, 1986). A relatively high incidence of LCs occurs in some parts of the hippocampal and olfactory systems, especially in the septum hippocampi and in the nucleus entopeduncularis; in the latter the LI% greatly exceeded those of all other regions of the brain (15.32%). It is noteworthy that both of these regions are derivatives of the ventral and Fig. 3 CELL PROLIFERATION IN THE BRAIN OF ADULT FROGS 467 Fig. 4. Photomicrographs of 3H-thymidine labelling in the ventricular wall. A,B: Transverse sections through the diencephalic recessus infundibularis. 3H-thymidine LCs are mainly located in the ependyma1 cell lining. B: A magnification showing one subependymal LC (arrow). Toluidine blue staining. Bars: 100 pm (A) and 20 pm (B). medial germinative zones of the telencephalon, in which residual proliferation in ependyma prevails over the dorsal zone in amphibians still at adult period of life. It is of interest that the corresponding homologous regions in mammalian brain (septum hippocampi, striatal complex) also show relatively active proliferation (Altman, 1966, 1969; Mareg and Lodin, 1974). In most other parenchymal regions, LCs were relatively small and appeared t o be glial cells; the LCs in the nucleus entopeduncularis were larger and looked like neurons. Also, in the septum hippocampi LCs Fig. 3. Photomicrographs of 3H-thymidine labelling in the ventricular wall. A,B Transverse section through the middle telencephalon. A Note the numerous 3H-thymidine LCs (mainly unipolar tanycytes) in the ependymal lining of the ventral part of the lateral ventricle. B Higher magnification of a detail in A. Some LCs are clearly visible in the ependymal lining. C,D: Transverse sections through the mesencephalic aqueductus Sylvii. In the ependymal lining of this localization LCs are much less numerous than in the ependyma of lateral ventricles. D: Higher magnification of a detail in C showing one 3Hthymidine LC in the ependymal lining. Toluidine blue staining. Bars: 100 pm (A,C) and 25 pm (B,D). might be neurons. Final classification of these cells is not possible in our material. Because of the pulse labelling schedule of our experiments, it is clear that labelling of neuron-like cells in our material is not due to migration of newly formed neurons from the adjacent germinative zones as described in non-mammalian vertebrates (Polenov et al., 1972; Raymond and Easter, 1983; Lopez-Garcia et al., 19881, or even some mammals (Kaplan and Bell, 1983). Finally, it should be pointed out that in the frog, as in mammals (MareS, 1986), LCs often occur in groups or patches of different size. This might be a consequence of repeated divisions of perennially active, or activated postmitotic cells. Functional and Comparative Comments The impact of cell division on number of brain cells in adults is not fully understood for any of the species studied. Cell division has been attributed mainly to cell renewal. Renewal of glial cells has been described in mice and rats (Korr, 1980; MareS and Lisy, 1980,1983), and in sexually mature monkeys (Eckenhoff and Rakic, 1988). We admit that cell division in the frog brain Fig. 5 CELL PROLIFERATION IN THE BRAIN OF ADULT FROGS 469 reflects cell renewal, especially in the regions in which vak Academy of Sciences and partially supported by the 3H-thymidine LI% is high and, at the same time, the Italian MURST and C.N.R. grants to G.B. pyknotic cells are present (Sturrock, 1979). However, LITERATURE CITED long lasting growth of cell populations, including neurons, has been found in non-mammals, e.g., in the optic Altman, J. 1963 Autoradiographic investigation of cell proliferation in the brain of rats and cats. Anat. Rec., 145.573-591. tectum of goldfish (Raymond and Easter, 1983) and Altman, J. 1966 Autoradiographic and histological studies of postnaamphibians (Gaze and Watson, 1968; Straznicky and tal neurogenesis. 11. A longitudinal investigation of the kinetics, Gaze, 1972). The cell proliferation observed in our migration and transformation of cells incorporating tritiated thymidine in infant rats, with special reference to postnatal neurostudy may also reflect the continuous growth of some genesis in some brain regions. J . Comp. Neurol., 128t431-473. parts of the brain. Altman, J. 1969 Autoradiographic and histological studies of postnaResidual proliferation in the brain of terrestrial vertal neurogenesis. IV. Cell proliferation and migration in the antebrates (Mareg, 1975; Kaplan and Bell, 1983; Lopezterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J . Comp. Neurol., 137t433-458. Garcia et al., 1988), including amphibians, displays evJ., and G.D. Das 1964 Autoradiographic examination of the ident features of homology, documented best in the Altman, effects of enriched environment on the rate of glial multiplication telencephalon, especially in the olfactory and hippoin adult rat brain. Nature, 204t1161-1163. campal regions. A long phylogenetic conservation of Bernocchi, G., E. Scherini, and V. Mare5 1985 Proliferazione cellulare nell’encefalo di rana adulta. Studio autoradiografico. Atti XX proliferation, or better, lesser disappearance of the Congr. SOC. It. Istoch., p. 160 (Abstract). ability to divide in ventral homologs of the brain is Capanna, E. 1969 Considerazioni sul telencefalo degli anfibi- Atti evident in humans (Kershman, 1939). The higher inAcc. Naz. Lincei (S. VIII), 9.5581. cidence of LCs in these parts of the brain lead us to Chauhan, A.N., and P.D. Lewis 1979 A quantitiative study of cell proliferation in ependyma and choroid plexus in the postnatal rat speculate on the influence of enhanced olfactory stimbrain. Neuropathol. Appl. Neurobiol., 5t303-309. ulation in terrestrial animals. In amphibians, the third Chetverukhin, V.K. 1978 Role of the preoptic recess ependyma in the olfactorial system and corticalization of the hippocamformation and physiologic regeneration of the nucleus praeoptipus developed as part of terrestrial adaptation (Claircus in Amphibians. In: Neurosecretion and Neuroendocrine Activity. Evolution, Structure and Function. W. Bargman, A. ambault, 1963; Capanna, 1969; Mazzi and Fasolo, Oksche, A. Polenov, and B. Scharrer, eds. Springer-Verlag, Ber1977). A possible relationship between division of glial lin, Heidelberg, pp. 145-151. cells and neural activity of the brain in some mammals Clairambault, P. 1963 Le telencephale de Discoglossus pictus (0th.). has been shown by 3H-thymidine autoradiography. Etude anatomique chez le tetard et chez l’adulte. J . Hirnforsch., 6237-121. Raising of rats in generally enriched or impoverished M.F., and P. Rakic 1988 Nature and fate of proliferative environments led to significant differences in the num- Eckenhoff, cells in the hippocampal dentate gyrus during life span of the bers of LCs in the occipital lobe of the forebrain (Altrhesus monkey. J. Neurosci. 8t2729-2747. man and Das, 1964). Rearing of rats in darkness re- Dalton, M.M., O.R. Hommes, and C.P. Leblond 1968 Correlation of glial proliferation with age in the mouse brain. J. Comp. Neurol., sulted in inhibition of cell division in the visual system 134r397-399. centers (MareS et al., 1977). DNA synthesis is reported Fleischauer, K. 1972 Ependyma and subependymal layer. In: The in activated neurons in vocalization centers of canaries Structure and Function of Nervous Tissue. Vol. IV. H. Bourne, ed. before the singing period (Goldman and Nottebohm, Academic Press, New York, pp. 1-46. 1983) and for neurons of the myenteric plexus during Gaze, R.M., and W.E. Watson 1968 Cell division and migration in the brain after optic nerve lesions. In: Ciba Foundation Symposium: experimental hypertrophy in rats (Giacobini-Robecchi Growth of the Nervous System. G.E.W. Wolstenholme and M. et al., 1985). A functional significance of cell division O’Connor, eds. Churchill Ltd, London, pp. 53-67. would agree with the lower incidence of LCs observed Giacobini-Robecchi, M.G., M. Cannas, and G. Filogamo 1985 Increase in the number and volume of myenteric neurons in the adult rat. in frogs during hibernation, since it is accompanied by Int. J. Dev. Neurosci., 3t673-675. a decrease in electrical activity of neurons in many Goldman, S.A., and F. Nottebohm 1983 Neuronal production migrabrain regions (Heller, 1979). Inhibition of proliferation tion and differentiation in a vocal control nucleus of the adult has also been observed in other mammalian organs female canary brain. Proc. Natl. Acad. Sci. USA, 8Ot2390-2394. during hibernation, and is considered to be a conse- Heller, H.C. 1979 Hibernation: neural aspects. Ann. Rev. Physiol., 41t305-321. quence of lower “requirement of new cells” rather than Hommes, O.R., and C.P. Leblond 1969 Mitotic division of neuroglia in a temperature-dependent decrease in activity of DNAnormal adult rat. J. Comp. Neurol., 129t269-278. synthesizing machinery (Kolaeva et al., 1980). Kaulan. . ,M.S.. and D.H. Bell 1983 Neuronal Droliferation in the 9ACKNOWLEDGMENTS This work is a part of the scientific joint program “Proliferation and differentiation of normal and tumor cells studied by cytochemical methods” between the Italian National Research Council and the Czechoslo- ~~ Fig. 5. Photomicrographs of 3H-thymidine labelling in the brain parenchyma. A , B Sagittal sections through the forebrain. B Higher magnification of a detail in A. Several LCs of neuronal type (arrows) are present in the nuclei of the septum hippocampi. C , D Transverse section through the preoptic area. D Higher magnification of the nucleus entopeduncularis showing a cluster of neuron-like 3H-thymidine LCs. Toluidine blue staining. Bars: 100 pm (A,C), 30 ym (B), and 15 pm (D). month-old rodent. Radioautographic studiof granule cells in the hippocampus. Exp. Brain Res., 52:l-5. Kaplan, M.S., and J.W. Hinds 1980 Gliogenesis of astrocytes and oligodendrocytes in the neocortical grey and white matter of the adult rat: Electron microscopic analysis of light radioautographs. J. Comp. Neurol., 193t711-727. Kershmam, J . 1939 Genesis of microglia in the human brain. Arch. Neurol. Psychiatr., 41 t24-50. Kirsche, W. 1967 Uber postembryonale Matrixzonen in Gehirn verschiedener Vertebraten und deren Beziehung zur Hirnbauplanlehre. Z. Mikrosk. Anat. Forsch., 77t313-406. Kolaeva, S.G., L.I. Kramarova, E.N. Ilyasova, and F.E. Ilyasov 1980 The kinetics and metabolism of the cells of hibernating animals during hibernation. Int. Rev. Cytol., 66t147-170. Korr, H. 1980 Proliferation of different cell types in the brain. Adv. Anat. Embryol. Cell Biol., 61:l-72. Kranz, D., and W. Richter 1970a Autoradiographische Untersuchungen uber die Lokalisation der Matrixzonen des Diencephalons von Juvenilen und Adulten Lebzstes retrculatus (Teleostei). Z. mikrosk. Anat. Forsch., 82t42-66. Kranz, D., and W. Richter 1970b Autoradiographische Untersuchun- 470 G. BERNOCCHI ET AL. gen zur DNA-Synthese im Cerebellum und in der Medulla oblongata von Teleostiern verschiedenen Lebensalters. Z. Mikrosk. Anat. Forsch., 82t264-292. Kranz, D., and W. Richter 1971 Autoradiographische Untersuchungen zur Regeneration des Tectum opticum von Lebistes reticulatus (Teleostei). Z. Mikrosk. Anat. Forsch., 84t420-428. Lopez-Garcia, C., A. Molowny, J.M. Garcia-Verdugo, and I. Ferrer 1988 Delayed postnatal neurogenesis in the cerebral cortex of lizards. Dev. Brain Res., 43t167-174. Mares, V. 1975 An autoradiographic study of regional differences in DNA synthesis in the brains of young adult mice. Acta Histochem., 53t70-76. Mares, V. 1986 DNA synthesis and cell number homeostasis in the brain. In: Role of RNA and DNA in Brain Function. A. Giuditta, B.B. Kaplan, and C. Zomzely-Neurath, eds. Martinus Nijhoff, Boston, pp. 247-255. Mares, V., and V. Liss 1980 Cell renewal and DNA turnover and radiolysis in the brain of young adult mice. A biochemical and autoradiographic study of 3H DNA synthesis and degradation. Physiol. Bohemoslov., 29:456 (Abstract). Mares, V., and V. Lisy 1983 Cell death in the dividing pool of cells in the mouse brain. Physiol. Bobemoslov., 32:385-392. Mares, V., G. Bruckner, T. Narovec, and D. Biesold 1977 The effect of different light regimes on DNA synthesis and cell division in the rat visual centres. Life Sci., 21t727-732. Mares. V.. and Z. Lodin 1974 An autoradioeraDhic studv of DNA synthesis in adolescent and adult mouse-foiebrain. Brain Res., 76557-561. . -. - - . - - -. MareS, V., Z. Lodin, and M. Jilek 1975 An estimate of the number of cells arising by division in mouse cerebral hemisphere from age one to 12 months: a n autoradiographic study of DNA synthesis. J. Comp. Neurol., 161t471-482. Mazzi, V., and A. Fasolo 1977 Neurologia Comparata. Boringhieri, Torino. Pannese, E. 1981The satellite cells of the sensory ganglia. Adv. Anat. Embryol. Cell Biol., 65:l-111. Paterson, J.A., A. Privat, E.A. Ling, and C.P. Leblond 1973 Investigation of glial cells in semithin sections 111. Transformation of subependymal cells into glial cells as shown by autoradiography after 3H Thymidine injection into lateral ventricle of the brain of young rats. J . Comp. Neurol., 149r83-102. Paul, E. 1967 Uber die Typen der Ependymzellen und ihre regionale Verteilung bei Rana temporaria L. Z. Zellforsch., 80r461-487. Polenov, A.L., V.K. Chetverukhin, and I.V. Jakovleva 1972 The role of the ependyma of the recessus praeopticus in formation and the physiological regeneration of the nucleus praeopticus in lower vertebrates. Z. Mikrosk. Anat. Forsch., 85.513-532. Rahmann, H. 1968 Autoradiographische Untersuchungen zum DNSstoffwechsel (Mitose-haufigkeit) im ZNS von Brachydanio rerio. J. Hirnforsch., 10:279-284. Raymond, P.A., and S.S. Easter 1983 Postembryonic growth of the optic tectum in goldfish. I. Location of germinal cells and numbers of neurons produced. J . Neurosci., 3t1077-1091. Reznikov, K.Ju. 1981 Proliferation of cells of the brain of vertebrates during normal development of the brain and its trauma. Publ. House Nauka, Moscow, pp. 1-149. Richter, W., and D. Kranz 1970a Die Abhangigkeit der DNS-synthese in den Matrixzonen des Mesencephalons vom Lebensalter der Versuchtiere, Lebistes reticulatus-Teleostei. Autoradiographische Untersuchungen. Z. Mikrosk. Anat. Forsch., 82t76-92. Richter, W., and D. Kranz 1970b Autoradiographische Untersuchungen uber die Abhangigkeit des 3H-Thymidin-Index vom Lebensalter in den Matrixzonen des Telencephalons von Lebistes reticulatus, Teleostei. Z. Mikrosk. Anat. Forsch., 81t530-554. Richter, W., and D. Kranz 1971 Altersabhangigkeit der Aktivitat der Matrixzonen im Gehirn von Xiphophorus helleri (Teleostei). Autoradiographische Untersuchungen. J. Hirnforsch., 13:109-115. Smart, I. 1961 The subependymal layer of the mouse brain and its cell production as shown by radioautography after thymidine -H3 injection. J . Comp. Neurol., 116:325-347. Smart, I., and C.P. Leblond 1961 Evidence for division and transformation of neuroglia cells in the mouse brain, as derived from radioautography after injection of thymidine-H3. J. Comp. Neurol., 116t349-367. Straznicky, K., and R.M. Gaze 1972 Development of the optic tectum in Xenopus laeuis: a n autoradiographic study. J. Embryol. Exp. Morphol., 26r87-115. Sturrock, R.R. 1979 A quantitative lifespan study of changes in cell number, cell division and cell death in various regions of the mouse forebrain. Neuropathol. Appl. Neurobiol., 5t433-456. Yanes Mendez, C., J.M. Martin Trujillo, M.A. Perez Batista, M. Monzon Mayor, and A. Marrero 1988 Ependymogenesis of the lizard basal areas. I. Ependymal zones. Z. Mikrosk. Anat. Forsch., 102555-572.