Delayed postnatal neurogenesis in the cerebral cortex of lizards

Developmental Brain Research, 43 (1988) 167-174 Elsevier

167

BRD 50804

Research Reports

Delayed postnatal neurogenesis in the cerebral cortex of lizards C. Lopez-Garcia 1, A. Molowny l, J.M. Garcia-Verdugo 1 and I. Ferrer 2 ICatedra de Citologia e Histologia, Facultad de Ciencias Biologicas, Universidad de Valencia. Burjasot, Valencia (Spain) and 2Unidad de Neuropatologia, Departamento de Anatomia Patologica. Hospital Principles de Espa~a, Hospitalet de Llobregat, Barcelona (Spain) (Accepted 11 May 1988)

Key words: Thymidine; Autoradiography; Reptile; Postnatal neurogenesis; Cerebral cortex; Hippocampus

Labelled cells were consistently observed in the medial cortex of the lizard brain after i.p. injections of tritiated thymidine (5 llCi/g b. wt. ), 1,7, 18 or 28 days of survival and posterior autoradiographic evaluation. In 3 groups of specimens (postnatal, young and adult) of the species Podarcis hispanica, after one day of survival, labelled cells were located in the ependymal cell layer underlying the medial cortex. After intermediate survival times (7, 18 days), labelled cells were found in 3 zones: the ependymal layer, the inner plexiform layer and the granular layer. After one month of survival, most labelled cells were observed in the granular layer. In the granular laver, these cells were distributed at random. These results show that postnatal neurogenesis in the medial cortex of the lizard occurs following a spatio-temporal pattern reminiscent of that found in the fascia dentata of the mammalian hippocampus. INTRODUCTION In the m a m m a l i a n brain, postnatal neurogenesis and recruitment of nerve cells, as revealed by means of tritiated thymidine labelling, have been consistently observed, among other specific regions, in the fascia dentata of the hippocampus in several species including the mouse 4, rat 1"2"s'28"52 and rhesus monkey 4v. Although a u t o r a d i o g r a p h i c analysis of neurogenesis in reptilian cortical structures has been carried out during embryonic d e v e l o p m e n t 22, no similar studies have been p e r f o r m e d on postnatal or adult lizards. However, previous morphological and quantitative studies have shown a significant increase in the number of nerve cells in the medial cortex of the lizard Podarcis hispanica during postnatal life 36. Furthermore, electron microscopical observations in adult specimens of Lacerta galloti have d e m o n s t r a t e d the presence of immature neurons attached to the vertical shafts of e p e n d y m o c y t e s in the inner plexiform layer of the medial cortex j9 thus suggesting that de-

layed neurogenesis and migration of neurons from the ventricular germinal centres may occur in the medial cortex of lizards throughout their life span. For this reason, we injected tritiated thymidine into postnatal, young and adult animals to check for the existence of labelled neurons in the medial cortex of the lizard Podarc& hispanica, by a u t o r a d i o g r a p h y of serial sections of the brain o b t a i n e d at different days after the injection of the D N A - r e p l i c a t i o n marker. MATERIALS AND METHODS Perinatal, young and adult specimens of the lizards species Podarcis hispanica (Steindachner, 1870) captured at the beginning of s u m m e r were used in this study. Perinatal specimens ( 1 - 3 0 days old) had 2 4 - 2 5 mm head-cloaca sizes and immature gonads. Young specimens (estimated age 6 - 1 2 months) had 30-35 mm sizes and m a t u r e gonads; they seemed not yet reproductive (females not yet fertilized). A d u l t speci-

Correspondence: C. Lopez-Garcia, Catedra de Citologia e Histologia, Facultad de Ciencias Biologicas, Universidad de Valencia. 46100 Burjasot, Spain. 0165-3806/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

168 mens (2-5 years old) had 40-55 mm sizes and mature

Electron microscopy

gonads; they were active in reproduction (i.e. fe-

A second sub-set of animals (4 weeks of survival) were perfused with 1.25% glutaratdehyde, 1'72 para-

males with fertilized eggs). Animals under semi-lethargic conditions (expo-

formaldehyde in 0.12 M phosphate buffer pH 7.2.

sure to 4 °C for 2 - 5 min) were subjected to a d a i h

After removal, the brains were immersed in the same

i.p. injection of tritiated thymidine (5/~Ci/g b. wt.)

fixative for 4--0 h. Transverse slices 3(10-40(/ /m~

during 3 consecutive days and then left in terrariums

thick were cut by a tissue chopper and collected in

simulating normal habitat conditions. The animals

phosphate buffer (0.12 M, pH 7.2) ~ith 8% glucose.

were sacrificed after different selected survival times

These slices were postfixed m 1%- osnlmm tetroxide,

(1,7, 18 and 28 days after the last injection).

7% glucose in phosphate buffer (0.12 M. pH "7 2t:

Light microscopy

some slices were e m b e d d e d in Araldite and others in Epon.

After ether anaesthesia, one set of animals were

Semithin sections were processed l:or autoradio-

intra-cardially perfused with formalin, the brain was

graphy as previously described (but exposed for two

extracted, immersed in Bouin fixative and e m b e d d e d

months at 4 :'C/. Then, sections ~ith labelled cells were re-embedded in Araldite or Epon and ultrathin sections of them were observed and photographed in

in paraffin. Cell counts were performed on 10-urn thick transverse complete serial sections of the brain. The slides bearing all the sections from each brain were dipped in autoradiographic emulsion (llford K-5 nuclear emulsion diluted 1:1 with water), dried in the dark and stored at 4 °C for 2 0 - 3 0 days. The slides were then immersed in Kodak D-19 developer for 5 - 8 rain, rinsed in water, fixed in 20% sodium thiosulfate, counterstained with Toluidine blue, dehydrated, cleared and mounted. Using a 100x objec-

a Jeol-IOI)S electron microscope. RESU LI:S

Labelled cells were consistently observed in pertnatal, young and adult specimens at all the survival

tive nuclei with a silver granule density exceeding 3 times that of the background were considered as

times after the injection of tritiated thymidine. In addition to other structures (e.g. the olfactory bulbs and the cerebellum), n u m b e r s of these cells were found in the medial cortex. No substantial n u m b e r s of labelled cells were e n c o u n t e r e d in the lateral, dor-

marked nuclei.

sal and dorsomedial cortices,

Fig. 1. Autoradiographically developed Nissl-stained transverse secnon at a caudal level of the telencephalon ol a perinatal specimen P. hispanica. One day of survival after their last dose of [3Hlthymidine. Note the presence of labelling in the ependyma arrows): MC. medial cortex: DMC. dorsomedial cortex. ( 145 x 1. Fig. 2. Higher magnification of ependyma from the same section as Fig. 1. 1450x ~. Fig. 3. Autoradiographically developed Nissl-stained transverse section at a caudal level of the telencephalon ot a young specimen ol P. hispanica. Twenty-eight days of survival after its last [3H]thymidinedose. Note the presence of labelling in both the cell layer of the medial cortex (MC) and in the ependyma: DMC. dorsomedial cortex. /130x L Fig. 4. Aspect of the inner plexiform layer (IPLI of the medial cortex in a transverse section l autoradiographically developed and Nissl-stained) of the telencephalon of an adult specimen with 7 days of survival after its last [3H]thymidinedose. Note the presence of some labelled cells in the IPL (arrows) as well as in the ependyma (EP) and in the cellular layer (CL). (400x Fig. 5. Higher magnification aspect of the IPL of the same section as Fig 4. Note the vertical fusiform shape of an unlahelled cell closc to a group of silver granules labelling another presumably similar cell. [ 1440x/. Fig. 6. Aspect of the ependyma (EP), inner plexiform layer (IPL) and cell layer (CLJ of the medial cortex ot a specimen of P. hispamca. Twenty-eight days of survival after its last [3H]thymidine injection. Note the presence of labelled cells both in the ependyma and m the cell layer. Young specimen ( 1310x ).

169

7%.

I

IPL

Q D

I

170 TABLE I Distribution of marked nuclei in the ependyma ( EPI. inner plexiJorm layer ¢IPL) and granular layer (GLI o! the medial cortex ot t'odarcis h 5panica

Numerical values given are percentages of all marked nuclei which were found in the medial cortex (survival times are those counted after the third daily dose of tritiated thymidine). Survival time

One day One week 18 days Four weeks

Perinatal

Young

Adult

EP

IPI,

GL

I51'

IPL

(3l,

['.f'

IPI.

(;1.

98 43 22 26

2 13 I1 ~

II 44 67 72

96 52 15 14

4 9 1 I

~/ 39 84 S5

98 69 8 '

2 14 {3 s

17 79 90

After one day of survival, most labelled cells in the cerebral cortex were located in the e p e n d y m a l region of the underlying medial cortex (Figs. 1, 2). A f t e r 28 days of survival most labelled cells in the cerebral cortex a p p e a r e d in the cell layer of the medial cortex (Fig. 3). A f t e r one week of survival, labelled cells were found in 3 zones of the medial cortex: the e p e n d y m a , the inner plexiform layer and the granular layer (Figs. 4, 5). In the inner plexiform layer, these cells had an elongated cellular body which was perpendicular to the pallial surface (Fig. 5). Labelled cells were also found in these 3 zones after 18 and 28 days of survival. In the granular layer, these cells were distributed at r a n d o m : no preferential distribution of labelled cells was observed, either near the inner plexiform or the o u t e r plexiform layer. H o w e v e r , striking quantitative differences were evident, which d e p e n d e d on the survival time. The n u m b e r of labelled cells progressively increased in the granular layer and decreased in the e p e n d y m a l layer as function of the survival time after the last injection of tritiated thymidine (see Figs. 1-5 and Table I). This spatio-temporal p a t t e r n was similar w h a t e v e r the age of the animals (see Table l). A f t e r one month of sur-

vival most labelled cells were found in the granular laver. In spite of these findings, some labelled cells were still present in the e p e n d y m a l laver 28 days after the cellular m a r k e r injection. This r e m a i n i n g population of labelled cells in the e p e n d y m a was particularly evident in perinatal and young specimens (Fig. 6L Electron m i c r o s c o p y

In the electron mtcroscope, the cell layer of the medial cortex of perinatal, young and adult specimens of P. hispanica, showed a similar a p p e a r a n c e to that found in other close lizard species ~s. Neuronal somata a p p e a r e d closely p a c k e d (Fig. 7); no g]ial cell somata could be d e t e c t e d intermingled with them All the labelled somata m the cell laver of the medial cortex tha! were observed in the electron microscope displayed similar m o r p h o l o g y ~o that of adjacent neuronal s o m a t a (Figs, 8, 9L i.e. well d e v e l o p e d nucleolus, dispersed chromatin, and Nissl substance. DISCUSSION The medial cortex in lizards is c o m p o s e d of densely packed neuronal somata with no glial elements be-

Fig. 7. Electron microscopic aspect of the cell layer of the medial cortex of an adult specimen of P. hispanica. Neuronal somata appem closely packed; nuclei have dispersed chromatine. Araldite (6000 x ). Fig. 8. One/~m semithin section of the cell layer of the medial cortex of an adult specimen 14 weeks of survival) processed for amoradiography. Some nuclei appear heavily labelled by silver granules. Epon embedding. ( t 900 × ). Fig. 9. Electron microscopic aspect of the cell somata numbered in Fig. 8 I rectangle). Labelled soma (3) has indistinct morphology to that of adjacent neuronal somata. Epon. (4100x).

171

172 tween them Is. Under normal conditions, astrocytes and microglial cells are almost absent. The glial component is restricted to the vertical varicose shafts of ependymocytes, which have their cellular body in contact with the ventricular wall, and oligodendrocytes lying in the supraependymal myelinic layer (alveus). Glial cells represent no more than 5c/~ of the total number of cells in the medial cortex of other close related species of lizards m w Although, in this study, a reduced number of labelled cells have been observed in the electron microscope, all of them displayed typical morphology of neuronal somata. So, labelled cells in this study have been considered to belong, in the largest proportion, to germinal cells, migrating neuroblasts I'~ and neurons. These criteria may be applied depending on their shape, size and position in the medial cortexV>5'( The results presented here show that the recruitment of nerve cells in the medial cortex of perinatal. young and adult specimens of the lizard Podarcis hispanica originates from the cellular division of germinal cells m the ependymat layer. Thus these findings corroborate previous observations on the postnatal increase in the number of nerve cells in the granular layer of the medial cortex of Podarcis hispanica >, as well as the postulated migration of neuroblasts ahmg the apical shafts of ependymocytic cells in the inner plexiform layer of the medial cortex m young specimens of Lacerta gallotil'( The cerebral cortex of lizards and the mammalian hippocampus have been considered homologous structures on the basis of their similar cytoarchitecture '~~a 15,30.38,3o,.41.4~.50, chemoarchitecture12.20.23.35.40,42 ~ a ~ connectivity m'323~ and ontogeny <>'-~:>'-~'. The coincidence of delayed postnatal neurogenesis in both the medial cortex of lizards and the fascia dentata of the hippocampus ~' adds new weight to the argument for the homology of these two structures. The guidance of neurons along the apical shafts of ependymocytes and the lack of any inside-outside gradient or vice versa further support the idea of either a convergence or a c o m m o n origin for the mechanisms regulating the neurogenetical processes in these cortical regions 15~'~4:. However, in lizards, a consistent pool of marked nuclei remains in the ependyma of all groups of ani-

mals for all the survival times studied, indicating the existence of a matrix or germinal zone > in the ependyma lining the medial cortex of lizards. The persistence of this pool m young and adult specimens, although suffering an apparent progressive decrease m older specimens I see Table II, may explain the capacity of delayed postnatal neurogenesis of this nervous center m lizards. The biological significance ol this finding remains unclear. In the cerebral cortex of lizards there ~s a system of Timm-posmve zones occupied by a specific kind ot Timm-reactive presynaptic boutons which have been compared to the projection zones of the mammalian hippocampal mossy fibres and wilh mossy fibre boutons. respectively 3<35>4". These Timm-positive zones undergo a consistent volumetric increase during perinatal, iuvenile and aduh lift: in the species P. hispanica I~. which is correlated with a parallel mcrease m the absolute numbers of Timm-reacfive boutons they contain 51. These Tnnm-reactive boutons seem to originate in neurons whose somata arc located in the cell laver of lhc medial cortex ~7 Whether the newlv formed neunms ot the cell laxm of the medial c o r t e x of lizards send axons and Timmreactive boulons It) the Timm-posinve zones remains a question open to further study. Recently, a reduced amount ot postnatally generated neurons has been detected m the mammalian hypothalamus ~a. In the case of non-mammalian vertebrates, delayed postnatal neurogcnesis has been extensively studied in the retina > > ~' and tectum opticum 4'~55'5:of adult fishes and amphibians. Postnatal neurogenesis has also been detected in cultures of fish spinal cord A(KNOWLE D(]EMENTS We thank Dr. Martinez-Guijarro for his help in performing the electron microscopic study. We thank Mr. P.J. Kanis for his help in correcting this manuscript and Dr. G. Danscher for his comments on it. This study has been partially supported by the Spanish C A I C y T and FISS.

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46

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