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Postnatal increase of GABA- and PV-IR cells in the cerebral cortex of the lizardPodarcis hispanica
BRAIN RESEARCH ELSEVIER
Brain Research 634 (1994) 168-172
Short Communication
Postnatal increase of GABA- and PV-IR cells in the cerebral cortex of the lizard Podarcis hispanica F.J. Martlnez-Guijarro *, J.M. Blasco-Ib~fiez, C. L6pez-Garcla Biologfa Celular, Facultad de Ciencias Biol6gicas. Universidad de Valencia, c/Dr. Molinev 50, 46100 Burjasot, Spain (Accepted 12 October 1993)
Abstract
The number and distribution of GABA- and parvalbumin (PV)-immunoreactive (IR) cells have been studied by immunocytochemistry in the cerebral cortex of newborn and adult lizards. The distribution of GABA-IR cells as well as that of PV-IR cells were similar in newborn and adult lizards, and PV-IR cells were GABA-IR in all cases. However, the absolute number of GABA- and PV-IR cells increased significantly during development. In addition, the rate of of GABA-IR cells also displaying PV immunoreactivity also increased after birth. Moreover, dendrites were rarely found to be PV-IR in newborn lizards, whereas they appeared stained in a Golgi-like manner in adult animals. These results suggest that the GABAergic neuronal population of the cerebral cortex of lizards experiments a significant increment in number and neurochemical maturation after birth.
Key words: GABA; Parvalbumin; Development; Lizard; Cerebral cortex
In the cerebral cortex of lizards, which has been correlated to the mammalian hippocampal formation [9], the neuronal population includes both principal cells and interneurons. The former are spiny projection neurons, and the latter are spine-free or sparsely spinous short axon neurons, most of them displaying GABA immunoreactivity [1334,19-21]. The calcium binding protein parvalbumin (PV) has been found to be a marker for most GABAergic calcium binding protein-containing cells in the cerebral cortex of lizards [14], and, as in the mammalian hippocampus and cerebral cortex, PV-IR cells have been demonstrated to establish both axosomatic and axoaxonic synaptic contacts on principal cells [6,7,15,24]. In the mammalian cerebral cortex and hippocampus, G A D - I R cells have been found to be generated prenatally [2,3,12,23]. However, PV expression in GABAergic cells has been described to appear and increase after birth [4,18,22]. G A B A and PV immunoreactivity patterns have been characterized in the cerebral cortex of adult lizards.
* Corresponding author. Fax: (34) (6) 386 4781. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) E 1 4 0 4 - Q
However, developmental data are completely lacking. The aim of this study was to analyze whether PV and G A B A immunoreactivities change in the cerebral cortex of lizards with postnatal development. Five adult lizards (47-50 mm snout-vent in length) and four newborn lizards (26-28 mm snout-vent in length) of the species Podarcis hispanica were used in this study. Animals were perfused through the heart under ether anaesthesia, first with saline (0.9% NaCI) for 1 min and then with 30 ml of fixative containing 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 (PB), for 30 min. Brains were removed and cut transversely at 50 p.m on a vibratome. After extensive washes in PB, free-floating adjacent vibratome sections from each brain were incubated to reveal different antigens, first in 10% normal goat serum for 45 min, and then in mouse anti-GABA (1 : 1000) [17] or mouse anti-PV (1 : 5000, Clone McAB 235) [5] for 2 days at 4°C. Thereafter, the sections were processed according to the peroxidase-antiperoxidase method. PB was used for washing and antiserum dilutions. The immunoperoxidase reaction was developed with 3,3'-diaminobenzidine as the chromogen. After immunostaining, the sections were treated with 0.5% OsO 4 for 30 min, dehydrated in ethanol, and flat-embedded in Durcupan ( A C / M Fluka). Triton X-100 was
169
F.J. Martfnez-Guijarro et al. / Brain Research 634 (1994) 168-172
PV
GABA ! " ,°
,',~ ~.......~.;.; ....... ~..~, ;o° .., ............... • .., .q °
:,~°
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°,
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LC
A
LC M
B
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MC I : ! ~ " -
........
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i:i;!" ":..' "'""'"""" D
Fig. 1. Diagrammatic drawings of the cerebral cortex of the lizard Podarcis hispanica at three different telencephalic levels: precommisural (i), commissural (ii) and postcommissural (iii), showing the distribution of immunoreactive cells for GABA and parvalbumin (PV) in an adult (A,B) and in a newborn lizard (C,D). Dots represent immunoreactiveperikarya cut in half at one surface of a 50/J.m section. DC, dorsal cortex; DMC, dorsomedial cortex; LC, lateral cortex; MC, medial cortex. Bar = 200/xm. excluded from the sera or washing solutions in order to limit the staining, as much as possible, to the surface of the sections. This improves identification of cut neurons for counting and for the 'mirror technique', used to analyze the coexistence of two different antigens in the same cell [8]. Control sections were processed in the same way except that the primary antibodies were omitted. No immunostaining was observed under control conditions. The number of cortical G A B A - and PV-IR cells per hemisphere were estimated as follows. The volume of the cortex of one hemisphere from each animal was first obtained. Then, the profiles of G A B A - I R cells cut in half on one of the surfaces of each 50 ~ m section
immunoreacted for G A B A were drawn with the aid of a camera lucida. They were counted and their diameter measured. The numerical density ( N v) was obtained for each animal according to Abercrombie's formula [1]. The number of G A B A - I R cells per hemisphere was calculated from N v and the cortical volume previously obtained. All PV-IR cells displayed G A B A immunoreactivity. Thus, the number of P V - I R cells in the dorsomedial and dorsal cortical areas could be estimated from the number of G A B A - I R cells in these areas and the rate of G A B A cells also displaying PV immunoreactivity (which was obtained using the 'mirror technique'). The number of P V - I R cells was estimated for the dorsome-
170
F.J. Martlnez-Guijarro et al. /Brain Research 634 (1994) 168-172
GABA
•
0"
o
opl
F.J. Mart[nez-Guijarro et al. / Brain Research 634 (1994) 168-172
dial and dorsal areas as a whole. The medial cortex was excluded because PV-IR cells are very rare, sometimes absent, in this cortical area. The lateral cortex was also excluded because no PV-IR cells were found in this area. The distribution of GABA-IR cells in the cortex of adult lizards was in agreement with earlier studies [14,15,20,21]. GABA-IR cells were found throughout the cerebral cortex (Figs. 1A and 2A) and the highest density of GABA-IR cell bodies in the medial, dorsomedial and dorsal cortical areas was found in the inner plexiform layer, followed by the outer plexiform layer. In the cell layer, GABA-IR cells were sparse and usually appeared near to the upper or lower rims of the layer. Regarding the lateral cortex, GABA-IR cells were mainly located in the outer plexiform layer and in the cell layer, but they were rare in the inner plexiform layer. In newborn lizards the distribution of GABA-IR cells (Figs. 1C and 2B) was similar to that found in adult animals. However, a significant increase in the number of GABA-IR cells from newborn to adult animals was found (Table 1). The cerebral cortex of lizards has been shown to undergo a remarkable postnatal development [11]. The number of cortical cells increases considerably during juvenile periods of life [10] and neuronal proliferation and migration have been described even in adult animals [9]. Thus, although there are no data about the time of origin of GABAergic cells in the cerebral cortex of lizards, the increase in the number of GABA-IR cells may be due to postnatal generation of at least part of the GABAergic neurons. Nevertheless, this possibility needs confirmation using double labelling for GABA and a cell proliferation marker. In adult lizards, the distribution of PV-IR cells (Figs. 1B and 2C) was similar to that previously described in this lizard species [14-16]. Most of the PV-IR cells appeared in the dorsomedial and dorsal cortices, they were rare in the medial cortex, and completely absent in the lateral cortex. PV-IR cells appeared concentrated in the inner plexiform layer, although they were also found in the outer plexiform layer and in the Cell layer. The PV-IR cells had long, vertically running dendrites penetrating all layers. Some cells had, in addition, long horizontally running branches in the inner plexiform layer. In newborn lizards (Figs. 1D and 2D), the distribution of PV-IR ceils was similar to that found in adult animals. However, it is interesting to note that PV immunoreactivity
171
Table 1 Number (per hemisphere) of GABA- and PV-IR cells in the cortex of adult and newborn lizards Specimen No. GABA cells (all cortical areas)
No. GABA cells (DMC + DC)
% G A B A / No. PV PV cells (DMC (DMC + DC) + DC)
Adult 1 2 3 4 5 .f +S.D.
12931 14713 13657 13126 13867 13658.8+701.4
9730 10467 9667 9721 10489
13.2 13.8 12.0 12.5 14.0
1285 1452 1172 1215 1468 1318.4+135.5
Newborn 1 2 3 4 £ +S.D.
7742 8411 7602 8805 8140+566.7
5237 6724 5278 6913
9.0 4.1 8.2 4.6
476 280 436 322 378.5-t-92.5
Differences in mean values for adult and newborn lizards were significant (P < 0.01) for Student's t-test. DC, dorsal cortex; DMC, dorsomedial cortex; % GABA/PV, rate of GABA-IR cells which are also PV-IR.
in dendrites was nearly absent in newborn lizards, being mostly restricted to cell bodies (Fig. 2D). Similar results have been found in the mammalian hippocampus, where a gradual increase of PV immunoreactivity in dendrites was observed, and has been suggested to correspond to a neurochemical and functional maturation of PV-containing ceils during hippocampal development [4,22]. The total number of PV-IR cells per hemisphere increases significantly from newborn to adult animals (Table 1). PV-IR cells were found to be GABA-IR both in adult [14,15] (Fig. 2E,F) and in newborn lizards (Fig. 2G,H). Thus, the increment in number of GABAIR cells described above may account for the increase of PV-IR cells. However, whether there is postnatal generation of PV-containing GABA-IR cells needs to be confirmed. Nevertheless, the rate of GABA-IR cells displaying PV immunoreactivity is higher in adult lizards than in newborn ones (Table 1). Therefore, factors other than the number increase of GABA-IR cells may contribute to the increment of PV-IR cells. In this respect, the emergence of PV expression during development is considered to be a marker of activation of circuits where PV-containing cells are involved [4,22]. Thus, the increase in the number of PV-IR cells found in the present study may be due to activation during
Fig. 2. A-D: survey of the dorsomedial cortex immunostained for GABA (A,B) or PV (C,D), in an adult (A,C) and in a newborn lizard (B,D). cl, cell layer; ipl, inner plexiform layer; opl, outer plexiform layer. Bar = 50/zm. E-F, G-H: photomicrograph of paired surfaces of two consecutive sections from an adult (E-F) and a newborn lizard (G-H) incubated for PV (E,G) or GABA (F,H). Arrows point to cell bodies cut in half showing immunoreactivity for both PV and GABA. Bar = 20/zm.
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development of GABAergic cells susceptible to express PV immunoreactivity. In summary, our results demonstrate a significant postnatal increase of GABA- and PV-IR cells in the cortex of lizards, and suggest a postnatal development and maturation of the inhibitory circuits where PV-IR cells are involved. The authors are grateful to Drs. M. Celio (University of Fribourg, Switzerland) and C. Matute (University of Pais Vasco, Bilbao, Spain) for gifts of antisera against PV, and GABA. This study was supported by grants from the Ram6n Areces Foundation, the Spanish DGICYT (PB90-0422) and IVEI. [1] Abercrombie, M., Estimation of nuclear population from microtome sections, Anat. Rec., 94 (1946) 239-247. [2] Amaral, D.G. and Kurz, J., The time of origin of cells demonstrating glutamic acid decarboxylase-like immunoreactivity in the hippocampal formation of the rat, Neurosci. Lett., 599 (1985) 33-39. [3] Bayer, S.A., Development of the hippocampal region in the rat. I. Neurogenesis examined with 3H-thymidine autoradiography, J. Comp. Neurol., 190 (1980) 87-114. [4] Bergmann, I., Nitsch, R. and Frotscher, M., Area-specific morphological and neurochemical maturation of non-pyramidal neurons in the rat hippocampus as revealed by parvalbumin immunocytochemistry, Anat. Embryol., 184 (1991) 403-409. [5] Celio, M.R., Baier, W., Scharer, L., de-Viragh, P.A. and Gerday, C., Monoclonal antibodies directed against the calcium binding protein parvalbumin, Cell Calcium, 9 (1988) 81-86. [6] De Felipe, J., Hendry, S.H. and Jones, E.G., Visualization of chandelier cell axons by parvalbumin immunoreactivity in monkey cerebral cortex, Proc. Natl. Acad. Sci. USA, 86 (1989) 2093-2097. [7] Kosaka, T., Katsumaru, H., Hama, K., Wu, J.-Y. and Heizmann, C.W., GABAergic neurons containing the Ca2+-binding protein parvalbumin in the rat hippocampus and dentate gyrus, Brain Res., 419 (1987) 119-130. [8] Kosaka, T., Kosaka, K., Tateishi, K., Hamaoka, Y., Yansihara, H., Wu, J.-Y. and Hama, K., GABAergic neurons containing CCK-8-1ike a n d / o r VIP-like immunoreactivities in the rat hippocampus and dentate gyrus, J. Comp. Neurol., 239 (1985) 420-430. [9] Lopez-Garcia, C., Molowny, A., Garcla-Verdugo, J.M. and Ferrer, I., Delayed postnatal neurogenesis in the cerebral cortex of lizards, Deu. Brain Res., 43 (1988) 167-174. [10] Lopez-Garcia, C., Tineo, P. and Del Corral, J., Increase of the neuron number in some cerebral cortical areas of a lizard, Podarcis hispanica (Steind; 1870) during postnatal periods of life, J. Hirnforsch., 25 (1984) 255-259. [11] L6pez-Garcla, C., Molowny, A., Rodrlguez-Serna, R., GarclaVerdugo, J.M. and Martlnez-Guijarro, F.J., Postnatal development of neurons in the telencephalic cortex of lizards. In W.K. Schwerdtfeger and W. Smeets (Eds.) The Forebrain of Reptiles:
Current Concepts of Structure and Function, Karger, Basel, 1988, pp. 122-130. [12] Liibbers, K., Wolff, J.R. and Frotscher, M., Neurogenesis of GABAergic neurons in the dentate gyrus: a combined autoradiographic and immunocytochemical study, Neurosci. Lett., 62 (1985) 317-322. [13] Martlnez-Guijarro, F.J., Desfilis, E. and Lopez-Garcia, C., Organization of the dorsomedial cortex in the lizard Podarcis hispanica. In W.K. Schwerdtfeger and P. Germroth (Eds.) The Forebrain in Non-Mammals. New Aspects of Structure and Det~elopment, Springer, Berlin, 1990, pp. 77-92. [14] Martlnez-Guijarro, F.J. and Freund, T.F., Distribution of GABAergic interneurons immunoreactive for calretinin, calbindin D28K and parvalbumin in the cerebral cortex of the lizard Podarcis hispanica, J. Comp. Neurol., 322 (1992) 449-461/. [15] Martlnez-Guijarro, F.J., Soriano, E., Del Rio, J.A., BlascoIb~ifiez, J.M. and L6pez-Garcla, C., Parvalbumin containing neurons in the cerebral cortex of the lizard Podarcis hispanica: morphology, ultrastructure and coexistence with GABA, somatostatin and neuropeptide Y, J. Comp. NeuroL, 336 (19931 447-467. [16] Martlnez-Guijarro, F.J., Soriano, E., Del Rio, J.A. and L6pezGarcla, C., Parvalbumin-immunoreactive neurons in the cerebral cortex of the lizard Podarcis hispanica, Brain Res., 547 (19911 339-343. [17] Mature, C. and Streit, P., Monoclonal antibodies demonstrating GABA-Iike immunoreactivity, Histochemistry, 86 (1986) 147157. [18] Nitsch, R., Bergmann, I., Kuppers, K., Mueller, G. and Frotscher, M., Late appearance of parvalbumin-immunoreactivity in the development of GABAergic neurons in the rat hippocampus, Neurosci. Lett., 118 (1990) 147-150. [19] Regidor, J., Martin-Trujillo, J.M., Lopez-Garcia, C. and MarinGuiron, F., Estudio citoarquitectonico de la corteza cerebral de reptiles. II. Tipologia dendritica y distribuci6n neuronal en areas corticales de Lacerta galloti, Trab. Inst. Cajal Inv. Biol., 66 (1974) 1-32. [20] Schwerdtfeger, W.K. and Lorente, M.J., Laminar distribution and morphology of gamma-aminobutyric acid (GABA)-immunoreactive neurons in the medial and dorsomedial areas of the cerebral cortex of the lizard Podarcis hispanica, J. Comp. Neurol., 278 (1988) 473-485. [21] Schwerdtfeger, W.K. and L6pez-Garcia, C., GABAergic neurons in the cerebral cortex of a lizard (Podarcis hispanica), Neurosci. Lett., 68 (1986) 117-121. [22] Solbach, S. and Cello, M.R., Ontogeny of the calcium binding protein parvalbumin in the rat nervous system, Anat. Embryol., 184 (1991) 103-124. [23] Soriano, E., Cobas, A. and Fair,n, A., Neurogenesis of glutamic acid decarboxylase immunoreactive cells in the hippocampus of the mouse. I: Regio superior and regio inferior, Z Comp. Neurol., 281 (1989) 586-602. [24] Soriano, E., Nitsch, R. and Frotscher, M., Axo-axonic chandelier cells in the rat fascia dentata: Golgi-electronmicroscopy and immunocytochemical studies, J. Comp. Neurol., 293 (1990) 1-25.
Brain Research 634 (1994) 168-172
Short Communication
Postnatal increase of GABA- and PV-IR cells in the cerebral cortex of the lizard Podarcis hispanica F.J. Martlnez-Guijarro *, J.M. Blasco-Ib~fiez, C. L6pez-Garcla Biologfa Celular, Facultad de Ciencias Biol6gicas. Universidad de Valencia, c/Dr. Molinev 50, 46100 Burjasot, Spain (Accepted 12 October 1993)
Abstract
The number and distribution of GABA- and parvalbumin (PV)-immunoreactive (IR) cells have been studied by immunocytochemistry in the cerebral cortex of newborn and adult lizards. The distribution of GABA-IR cells as well as that of PV-IR cells were similar in newborn and adult lizards, and PV-IR cells were GABA-IR in all cases. However, the absolute number of GABA- and PV-IR cells increased significantly during development. In addition, the rate of of GABA-IR cells also displaying PV immunoreactivity also increased after birth. Moreover, dendrites were rarely found to be PV-IR in newborn lizards, whereas they appeared stained in a Golgi-like manner in adult animals. These results suggest that the GABAergic neuronal population of the cerebral cortex of lizards experiments a significant increment in number and neurochemical maturation after birth.
Key words: GABA; Parvalbumin; Development; Lizard; Cerebral cortex
In the cerebral cortex of lizards, which has been correlated to the mammalian hippocampal formation [9], the neuronal population includes both principal cells and interneurons. The former are spiny projection neurons, and the latter are spine-free or sparsely spinous short axon neurons, most of them displaying GABA immunoreactivity [1334,19-21]. The calcium binding protein parvalbumin (PV) has been found to be a marker for most GABAergic calcium binding protein-containing cells in the cerebral cortex of lizards [14], and, as in the mammalian hippocampus and cerebral cortex, PV-IR cells have been demonstrated to establish both axosomatic and axoaxonic synaptic contacts on principal cells [6,7,15,24]. In the mammalian cerebral cortex and hippocampus, G A D - I R cells have been found to be generated prenatally [2,3,12,23]. However, PV expression in GABAergic cells has been described to appear and increase after birth [4,18,22]. G A B A and PV immunoreactivity patterns have been characterized in the cerebral cortex of adult lizards.
* Corresponding author. Fax: (34) (6) 386 4781. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) E 1 4 0 4 - Q
However, developmental data are completely lacking. The aim of this study was to analyze whether PV and G A B A immunoreactivities change in the cerebral cortex of lizards with postnatal development. Five adult lizards (47-50 mm snout-vent in length) and four newborn lizards (26-28 mm snout-vent in length) of the species Podarcis hispanica were used in this study. Animals were perfused through the heart under ether anaesthesia, first with saline (0.9% NaCI) for 1 min and then with 30 ml of fixative containing 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 (PB), for 30 min. Brains were removed and cut transversely at 50 p.m on a vibratome. After extensive washes in PB, free-floating adjacent vibratome sections from each brain were incubated to reveal different antigens, first in 10% normal goat serum for 45 min, and then in mouse anti-GABA (1 : 1000) [17] or mouse anti-PV (1 : 5000, Clone McAB 235) [5] for 2 days at 4°C. Thereafter, the sections were processed according to the peroxidase-antiperoxidase method. PB was used for washing and antiserum dilutions. The immunoperoxidase reaction was developed with 3,3'-diaminobenzidine as the chromogen. After immunostaining, the sections were treated with 0.5% OsO 4 for 30 min, dehydrated in ethanol, and flat-embedded in Durcupan ( A C / M Fluka). Triton X-100 was
169
F.J. Martfnez-Guijarro et al. / Brain Research 634 (1994) 168-172
PV
GABA ! " ,°
,',~ ~.......~.;.; ....... ~..~, ;o° .., ............... • .., .q °
:,~°
'..i
°,
,° ,° I ° *~..'...'~.~°
~.- .:'..
M ~
LC
A
LC M
B
~"
MC I : ! ~ " -
........
•
, ::...
;'~'.ilLe
"....
."'.....:.'." ". • "::::~ r ; " i ' " . . . . . : r ;".:~'" ":~' . .o'. .;:.,..-'°,~° .°.°°°°
i:i;!" ":..' "'""'"""" D
Fig. 1. Diagrammatic drawings of the cerebral cortex of the lizard Podarcis hispanica at three different telencephalic levels: precommisural (i), commissural (ii) and postcommissural (iii), showing the distribution of immunoreactive cells for GABA and parvalbumin (PV) in an adult (A,B) and in a newborn lizard (C,D). Dots represent immunoreactiveperikarya cut in half at one surface of a 50/J.m section. DC, dorsal cortex; DMC, dorsomedial cortex; LC, lateral cortex; MC, medial cortex. Bar = 200/xm. excluded from the sera or washing solutions in order to limit the staining, as much as possible, to the surface of the sections. This improves identification of cut neurons for counting and for the 'mirror technique', used to analyze the coexistence of two different antigens in the same cell [8]. Control sections were processed in the same way except that the primary antibodies were omitted. No immunostaining was observed under control conditions. The number of cortical G A B A - and PV-IR cells per hemisphere were estimated as follows. The volume of the cortex of one hemisphere from each animal was first obtained. Then, the profiles of G A B A - I R cells cut in half on one of the surfaces of each 50 ~ m section
immunoreacted for G A B A were drawn with the aid of a camera lucida. They were counted and their diameter measured. The numerical density ( N v) was obtained for each animal according to Abercrombie's formula [1]. The number of G A B A - I R cells per hemisphere was calculated from N v and the cortical volume previously obtained. All PV-IR cells displayed G A B A immunoreactivity. Thus, the number of P V - I R cells in the dorsomedial and dorsal cortical areas could be estimated from the number of G A B A - I R cells in these areas and the rate of G A B A cells also displaying PV immunoreactivity (which was obtained using the 'mirror technique'). The number of P V - I R cells was estimated for the dorsome-
170
F.J. Martlnez-Guijarro et al. /Brain Research 634 (1994) 168-172
GABA
•
0"
o
opl
F.J. Mart[nez-Guijarro et al. / Brain Research 634 (1994) 168-172
dial and dorsal areas as a whole. The medial cortex was excluded because PV-IR cells are very rare, sometimes absent, in this cortical area. The lateral cortex was also excluded because no PV-IR cells were found in this area. The distribution of GABA-IR cells in the cortex of adult lizards was in agreement with earlier studies [14,15,20,21]. GABA-IR cells were found throughout the cerebral cortex (Figs. 1A and 2A) and the highest density of GABA-IR cell bodies in the medial, dorsomedial and dorsal cortical areas was found in the inner plexiform layer, followed by the outer plexiform layer. In the cell layer, GABA-IR cells were sparse and usually appeared near to the upper or lower rims of the layer. Regarding the lateral cortex, GABA-IR cells were mainly located in the outer plexiform layer and in the cell layer, but they were rare in the inner plexiform layer. In newborn lizards the distribution of GABA-IR cells (Figs. 1C and 2B) was similar to that found in adult animals. However, a significant increase in the number of GABA-IR cells from newborn to adult animals was found (Table 1). The cerebral cortex of lizards has been shown to undergo a remarkable postnatal development [11]. The number of cortical cells increases considerably during juvenile periods of life [10] and neuronal proliferation and migration have been described even in adult animals [9]. Thus, although there are no data about the time of origin of GABAergic cells in the cerebral cortex of lizards, the increase in the number of GABA-IR cells may be due to postnatal generation of at least part of the GABAergic neurons. Nevertheless, this possibility needs confirmation using double labelling for GABA and a cell proliferation marker. In adult lizards, the distribution of PV-IR cells (Figs. 1B and 2C) was similar to that previously described in this lizard species [14-16]. Most of the PV-IR cells appeared in the dorsomedial and dorsal cortices, they were rare in the medial cortex, and completely absent in the lateral cortex. PV-IR cells appeared concentrated in the inner plexiform layer, although they were also found in the outer plexiform layer and in the Cell layer. The PV-IR cells had long, vertically running dendrites penetrating all layers. Some cells had, in addition, long horizontally running branches in the inner plexiform layer. In newborn lizards (Figs. 1D and 2D), the distribution of PV-IR ceils was similar to that found in adult animals. However, it is interesting to note that PV immunoreactivity
171
Table 1 Number (per hemisphere) of GABA- and PV-IR cells in the cortex of adult and newborn lizards Specimen No. GABA cells (all cortical areas)
No. GABA cells (DMC + DC)
% G A B A / No. PV PV cells (DMC (DMC + DC) + DC)
Adult 1 2 3 4 5 .f +S.D.
12931 14713 13657 13126 13867 13658.8+701.4
9730 10467 9667 9721 10489
13.2 13.8 12.0 12.5 14.0
1285 1452 1172 1215 1468 1318.4+135.5
Newborn 1 2 3 4 £ +S.D.
7742 8411 7602 8805 8140+566.7
5237 6724 5278 6913
9.0 4.1 8.2 4.6
476 280 436 322 378.5-t-92.5
Differences in mean values for adult and newborn lizards were significant (P < 0.01) for Student's t-test. DC, dorsal cortex; DMC, dorsomedial cortex; % GABA/PV, rate of GABA-IR cells which are also PV-IR.
in dendrites was nearly absent in newborn lizards, being mostly restricted to cell bodies (Fig. 2D). Similar results have been found in the mammalian hippocampus, where a gradual increase of PV immunoreactivity in dendrites was observed, and has been suggested to correspond to a neurochemical and functional maturation of PV-containing ceils during hippocampal development [4,22]. The total number of PV-IR cells per hemisphere increases significantly from newborn to adult animals (Table 1). PV-IR cells were found to be GABA-IR both in adult [14,15] (Fig. 2E,F) and in newborn lizards (Fig. 2G,H). Thus, the increment in number of GABAIR cells described above may account for the increase of PV-IR cells. However, whether there is postnatal generation of PV-containing GABA-IR cells needs to be confirmed. Nevertheless, the rate of GABA-IR cells displaying PV immunoreactivity is higher in adult lizards than in newborn ones (Table 1). Therefore, factors other than the number increase of GABA-IR cells may contribute to the increment of PV-IR cells. In this respect, the emergence of PV expression during development is considered to be a marker of activation of circuits where PV-containing cells are involved [4,22]. Thus, the increase in the number of PV-IR cells found in the present study may be due to activation during
Fig. 2. A-D: survey of the dorsomedial cortex immunostained for GABA (A,B) or PV (C,D), in an adult (A,C) and in a newborn lizard (B,D). cl, cell layer; ipl, inner plexiform layer; opl, outer plexiform layer. Bar = 50/zm. E-F, G-H: photomicrograph of paired surfaces of two consecutive sections from an adult (E-F) and a newborn lizard (G-H) incubated for PV (E,G) or GABA (F,H). Arrows point to cell bodies cut in half showing immunoreactivity for both PV and GABA. Bar = 20/zm.
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