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Asynchronism in the neurogenesis of GABAergic and non-GABAergic neurons in the mouse hippocampus
Developmental Brain Research, 30 (1986) 88-t)2 Elsevier
88 BRD60165
Asynchronism in the neurogenesis of GABAergic and non-GABAergic neurons in the mouse hippocampus* EDUARDO SORIANO 1, ALBERTO COBAS2 and ALFONSO FAIREN2'3 1Departamento de Morfolog(a Microsc6pica, Facultad de Biologia, Universidad de Barcelona, Barcelona and 2Unidad de Neuroanatomia, lnstituto Cajal, CSIC, Madrid, (Spain) and 31NSERM U. 106, Suresnes, (France)
(Accepted 3 June 1986) Key words." Glutamic acid decarboxylase immunocytochemistry - - v-Aminobutyric acid (GABA)ergic neurons - Neurogenesis - - [3H]thymidine autoradiography - - Hippocampus - - Mouse
The time course of the neurogenesis of cells showing glutamic acid decarboxylase (GAD) immunoreactivity has been analyzed in the dorsal hippocampus and area dentata of the mouse. The quantitative data indicate that y-aminobutyric acid (GABA)ergic cells are generated prenatally, and that their peaks of neurogenesis occur before the peaks of neurogenesis of non-GABAergic cells. Additionally, GABAergic cells in the hippocampus proper follow a 'sandwich' sequence of positioning. In the neocortex of the mouse, the timing of neurogenesis of GAD-positive cells does not differ essentially from that of non-immunoreactive ( n o n - G A D ) cells 7. Both types of cells follow an inside-out generation sequence (see also ref. 14). Their periods of neurogenesis overlap, although the peaks are not exactly synchronous ( E l 4 for the GAD-positive cells; E l 3 for the n o n - G A D ones) 7. To explore whether a similar overlapping also occurs in other cortical structures, we analyzed the time course of prenatal neurogenesis of GAD-positive and n o n - G A D cells in the dorsal hippocampus of mice. Here, we show quantitative data indicating that GAD-positive neurons are generated prenatally, and that their peaks of autoradiographic labeling occur before the ones corresponding to n o n - G A D cells. The material of this experiment was partially the same as that used in the above-mentioned study 7. Briefly, pregnant C57B1 mice (Iffa Credo, Lyon, France) received single i.p. injections of 5/~Ci/g of [3H]thymidine (30 Ci/mMol, C . E . A . , Saclay, France) between embryonic days E l 0 and El7. Two pregnant females were injected at E l 0 , 13, 14 or 17, and one at E l l , 12, 15 or 16. Mating day, ascer-
tained by the presence of a vaginal plug, was considered as E0. Offspring were sacrificed at 3 months of age. From each litter, two animals were used in the present experiment. Under chloral hydrate anesthesia, the animals received a bilateral injection Of ]/~1 colchicine 16 in saline (20/~g//d) in the cortex overlying the dorsal hippocampus. After a survival time of 24 h they were perfused, under ether anesthesia, with p e r i o d a t e - l y s i n e - 4 % paraformaldehyde fixative 13. Coronal 20-#m-thick sections were cut with a freezing microtome and processed for G A D immunocytochemistry using the P A P method 22. The characteristics of the anti-GAD antibody (courtesy of Dr. M.L. Tappaz, Lyon, France) have been reported elsewhere 15. Triton X-100 was used to increase penetration of the immunoreagents. Sections were mounted on chrome-alum gelatinized slides and air dried. The sections were defatted, dipped in NTB-2 and exposed at 4 °C for one month before development in D19. Examples of [3H]thymidine-labeled GAD-positive cells are shown in Fig. 1. The clusters of silver grains (arrows) over unstained structures were interpreted to label n o n - G A D neurons. The possibility that the
* A preliminary report of this work has been published (ref. 21). Correspondence: E. Soriano, Departamento de Morfologia Microsc6pica, Facultad de Biologia, Universidad de Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain 0165-3806/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
Fig. 1. Illustrations of [3H]thymidine-labeled GAD-positive and non-GAD cells. Nomarski photomicrographs. Bar = 25/~m. A: two GAD-positive cells, probably pyramidal basket cells, located in the deep aspect of the granular layer (SG) of area dentata. Both were heavily labeled by [3H]thymidine after a pulse injection at El4. H, hilus. B: a multipolar GAD-positive cell located in the inferior zone of the stratum moleculare (SM) of area dentata. It became labeled after a pulse at El4. An arrow points to a cluster of silver grains labeling a non-GAD cell in stratum granulosum (SG). C: a GAD-positive cell in stratum radiatum (SR) of regio inferior, labeled by [3H]thymidine at E13. A cluster of silver grains (arrow) marks the presence of a non-GAD cell in stratum pyramidale (SP). D: two GAD-positive cells in the inferior aspect of the stratum pyramidale of regio inferior. One of them was labeled by [3H]thymidine at El3. E: [3H]thymidine-labeled cells in the inferior aspect of stratum pyramidale of regio superior after a pulse at El4. One of them is GAD-positive. Arrows point to labeled non-GAD cells.
90 clusters were associated to glial cells was ruled out (see refs. 7 and 19 for discussion). The positions of labeled cells were plotted onto camera lucida charts. Per injection day, a total of 32 such charts were made for GAD-positive cells, and 8 for n o n - G A D cells. The charts were taken from at least two littermates labeled at any given embryonic day. Animals from two different litters were used in the case of E l 3 , E l 4 and E l 7 injections. Numerical densities (number of labeled cells per m m 2) were calculated. In each major anatomical subdivison (area dentata, regio inferior and regio superiorS), the distributions of labeled GAD-positive and n o n - G A D cells (numerical densities vs. embryonic days, see Fig. 2) were analyzed using one-way analysis of variance and Tukey's multiple comparison test 24. The first [3H]thymidine-labeled n o n - G A D cells were observed after E l l injections. These labeled cells were present, in very small numbers, in all subdivisions of the hippocampus. The evolution of the labeling can be followed in Fig. 2 (dashed lines). In area dentata, the labeling of n o n - G A D cells increased steadily during the whole embryonic period studied. Most of these n o n - G A D cells were located in the granular layer and must correspond to granule cells, of which a large proportion is generated postnatally L3'4'17. On the other hand, n o n - G A D cells in the hilus, many of which correspond to modified pyramids, were labeled between E12 and El4. In the hippocampus proper, no major differences were found with previous autoradiographic analyses in the mouse 3,6. Thus, in the regio inferior, n o n - G A D cells reached a peak of labeling at E l 4 and El5. In the regio superior, the maximum labeling of n o n - G A D cells started at E l 5 and lasted until El6. A regio inferior-regio superior gradient, as defined in previous studies 3'4']8, is evident (Fig. 2). In area dentata, GAD-positive cells (Fig. 2, continuous line) reached their maximum labeling between E l 3 and E l 4 , and showed a clear decline during successive days of injection. After pulses at E l 7 , only a few GAD-positive cells became labeled. This labeling pattern is compatible with the idea that GAD-positive cells in area dentata are generated prenatally 2,~°. In the hippocampus proper, maximum labeling of GAD-positive cells started earlier than in area dentata. In regio inferior, this maximum was reached al-
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Fig. 2. Mean numerical densities (number of cells per mm2) and S.E.M.s of radioactively labeled cells at Ell-E17. Non-GAD cells, dashed lines; GAD-positive cells, continuous lines. Data were obtained from camera lucida charts. Area dentata: the labeling of non-GAD cells increases steadily during the embryonic period analyzed. A maximum of GAD-positive cell labeling occurs at El3 and El4. Differences exist between El4 and El2, and between El4 and El5 (P < 0.01). Regio inferior: the maximum of non-GAD cell labeling occurs at El4 and El5 (P < 0.01). GAD-positive cells reach their maximum labeling at El2 and El3 (P < 0.01). Regio superior: the maximum labeling of non-GAD cells is observed after injections at El5 and El6. Significant differences are found between El5 and El4 (P < 0.05) and between El6 and El7 (P < 0.01). The highest GAD-positive cell labeling is found between El2 and El5. Differences exist between El2 and Ell (P < 0.01) and between El5 and El6 (P < 0.05).
ready at E12 and E l 3 but decreased abruptly thereafter, reaching zero levels at El6. In regio superior, the maximum labeling occurred from E l 2 through
91
Fig. 3. Camera lucida drawings of the dorsal hippocampus showing the locations of GAD-positive cells labeled by [3H]thymidine at E12-E16. The cells found in 4 adjacent sections were pooled together onto each single drawing. See text for details. RI, regio inferior; RS, regio superior; AD, area dentata.
El5, and very few GAD-positive cells became labeled at E16 and E17. Similarly to GAD-negative cells, GAD-positive cells followed a regio inferiorregio superior gradient of neurogenesis. We conclude that, in the hippocampus proper, the peaks of neurogenesis occur earlier for the GAD-positive than for the GAD-negative cells. In both hippocampal subdivisions, but especially in the regio superior, GAD-positive cells showed a distinctive radial gradient (sandwich gradient), illus-
trated in Fig. 3. Injections at E l 2 resulted in the labeling of GAD-positive cells in the stratum oriens (close to the alveus), in the upper part of the stratum radiatum, and in the interface between the latter and the stratum lacunosum-moleculare. Injections at El3 resulted in a widespread distribution of labeled neurons among all layers of the hippocampus, including the stratum pyramidale. Injections from E l 4 onwards labeled GAD-positive cells in the stratum pyramidale and in the adjacent parts of the stratum radiatum and stratum oriens. This sandwich gradient has been detected previously in conventional autoradiographic studies 4'8, but the present results suggest that it is mainly related to the differentiation of GAD-positive cells. A detailed account on neurogenetic gradients of GAD-positive cells in the mouse hippocampus is in preparation. It is generally accepted that most hippocampal GABAergic neurons are interneurons 16, although there may be some exceptions z°. The vast majority of the non-GABAergic neurons (pyramidal and granule cells) are projection neurons. Thus, and contrarily to the generally accepted time sequence of neurogenesis 9, in the hippocampus the local circuit neurons are born earlier than the projection neurons. By combining [3H]thymidine autoradiography and immunocytochemistry, we were able to visualize the particular spatiotemporal gradients of generation of the GAD-immunoreactive neuron population which otherwise could be masked by the major neuronal types of the same region. The reasons for the differences with neocortical neurogenesis 7'14 are still unclear. It is in the marginal and intermediate zones of the developing neocortex where the first postmitotic cells can be recognized 11"12, and where the earliest GABAergic cells are identifiable 23. Differences become less conspicuous if we compare the adult hippocampus with the postnatal neocortex, before massive cell death reported for the marginal and intermediate zones of neocortex la has occurred. We thank C. Sotelo and M. Wassef for their advice, M.L. Tappaz for providing the anti-GAD antibody, and J.P. Rio, F. Roger and J. Simmons for technical help. Supported by the Fondation pour la Recherche M6dicale (France), CNRS (France) and CSIC/CAICYT (Spain), Grant 154/1985.
92 1 Altman, J. and Das, G., Autoradiographic and histological evidence of postnatal neurogenesis in rats, J. Comp. NeuroI., 124 (1965) 319-336. 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., 59 (1985) 33-39. 3 Angevine, J.B., Jr., Time of neuron origin in the hippocampal region: an autoradiographic study in the mouse, Exp. Neurol., Suppl. 13 (1965) 1-70. 4 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. 5 Blackstad, T.W., Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination, J. Comp. Neurol., 105 (1956) 417-538. 6 Caviness, V.S., Jr., Time of neuron origin of the hippocampus and dentate gyrus of normal and reeler mutant mice: an autoradJographic analysis, J. Comp. Neurol., 151 (1973) 113-120. 7 Fair6n, A., Cobas, A. and Fonseca, M., Times of generation of glutamic acid decarboxylase immunoreactive neurons in mouse somatosensory cortex, J. Comp. Neurol., in press. 8 Hine, R.J. and Das, G.D., Neuroembryogenesis in the hippocampal formation of the rat. An autoradiographic study Z. Anat. Entwickl.-Gesch., 144 (1974) 173-186. 9 Jacobson, M., Developmental Neurobiology, Plenum, New York, 1978, 562 pp.. 10 LUbbers, K., Wolff, J.R. and Frotscher, M., Neurogenesis of GABAergic neurons in the rat dentate gyrus: a combined autoradiographic and immunocytochemical study, Neurosci. Lett., 62 (1985) 317-322. 11 Luskin, M.B. and Shatz, C.J., Studies of the earliest generated cells of the cat's visual cortex: cogeneration of subplate and marginal zones, J. Neurosci., 5 (1985) 1062-1075. 12 Marfn-Padilla, M., Early prenatal ontogenesis of the cerebral cortex (neocortex) of the cat (Felis domestica). A Golgi study. I. The primordial neocortical organization, Z. Anat. Entwickl.-Gesch., 134 (1971) 117-145. 13 McLean, I.W. and Nakane, P.K., Periodate lysine-paraformaldehyde fixative. A new fixative for immuno-electron
14
15
16
17
18
19
20
21
22
23
24
microscopy, J. Histochem. Cytochem., 22 (1974) 1077-1083. Miller, M.W., Cogeneration of retrogradely labeled corticocortical projection and GABA-immunoreactive local circuit neurons in cerebral cortex. Dev. Brain Res., 23 (1985) 187-192. Oertel, W.H., Schmechel, D.E., Tappaz, M.L. and Kopin, I.J., Production of a specific antiserum to cat brain glutamic acid decarboxylase by injection of an antigen-antibody complex, Neuroscience. 6 (1981) 2689-2700. Ribak, C.E., Vaughn, J.E. and Saito, K., Immunocytochemical localization of glutamic acid decarboxytase in neuronal somata following colchicine inhibition of axonal transport, Brain Res., 140 (1978) 315-332. Schlessinger, A.R., Cowan, W.M. and Gottlieb, D.I., An autoradiographic study of the time of origin and the pattern of granule cell migration in the dentate gyrus of the rat, J. Comp. Neurol., 159 (1975) 149-176. Schlessinger, A.R., Cowan, W.M. and Swanson, L.W., The time of origin of neurons in Ammon's horn and the associated retrohippocampal fields, Anat. Embryol., 154 (1978) 153-173. Schmechel, D.E. and Rakic, P., A Golgi study of radial glial cells in developing monkey telencephalon: morphogenesis and transformation into astrocytes, Anat. Ernbrvol., 156 (1979) 115-152. Seress, L. and Ribak, C.E., GABAergic cells in the dentate gyrus appear to be local circuit and projection neurons, Exp. Brain Res., 50 (1983) 173-182. Soriano, E., Cobas. A., Tappaz, M.L. and Fair6n. A., Times of origin of GAD positive neurons in the mouse hippocampus and area dentata, Neurosci. Lett., Suppl. 22 (1985) $330 (Abstract).. Sternberger, L.A., Hardy, P.H., Cuculis, J.J. and Meyer, H.G., The unlabeled antibody method of immunohistochemistry. Preparation and properties of soluble antigen complex, and its use in identification of spirochetes, J. Histochem. Cytochem., 18 (1970) 315-333. Wolff, J.R., Bottcher, H., Zetzsche. T., Oertel, W.H. and Chronwall, B.M., Development of GABAergic neurons it, rat visual cortex as identified by glutamate decarboxylaselike immunoreactivity, Neurosci. Lett., 47 (1984) 207-212. Zar, J.H., Biostatistical analysis, Prentice-Hall, Englewood Cliffs, 1984, pp. 163,186.
88 BRD60165
Asynchronism in the neurogenesis of GABAergic and non-GABAergic neurons in the mouse hippocampus* EDUARDO SORIANO 1, ALBERTO COBAS2 and ALFONSO FAIREN2'3 1Departamento de Morfolog(a Microsc6pica, Facultad de Biologia, Universidad de Barcelona, Barcelona and 2Unidad de Neuroanatomia, lnstituto Cajal, CSIC, Madrid, (Spain) and 31NSERM U. 106, Suresnes, (France)
(Accepted 3 June 1986) Key words." Glutamic acid decarboxylase immunocytochemistry - - v-Aminobutyric acid (GABA)ergic neurons - Neurogenesis - - [3H]thymidine autoradiography - - Hippocampus - - Mouse
The time course of the neurogenesis of cells showing glutamic acid decarboxylase (GAD) immunoreactivity has been analyzed in the dorsal hippocampus and area dentata of the mouse. The quantitative data indicate that y-aminobutyric acid (GABA)ergic cells are generated prenatally, and that their peaks of neurogenesis occur before the peaks of neurogenesis of non-GABAergic cells. Additionally, GABAergic cells in the hippocampus proper follow a 'sandwich' sequence of positioning. In the neocortex of the mouse, the timing of neurogenesis of GAD-positive cells does not differ essentially from that of non-immunoreactive ( n o n - G A D ) cells 7. Both types of cells follow an inside-out generation sequence (see also ref. 14). Their periods of neurogenesis overlap, although the peaks are not exactly synchronous ( E l 4 for the GAD-positive cells; E l 3 for the n o n - G A D ones) 7. To explore whether a similar overlapping also occurs in other cortical structures, we analyzed the time course of prenatal neurogenesis of GAD-positive and n o n - G A D cells in the dorsal hippocampus of mice. Here, we show quantitative data indicating that GAD-positive neurons are generated prenatally, and that their peaks of autoradiographic labeling occur before the ones corresponding to n o n - G A D cells. The material of this experiment was partially the same as that used in the above-mentioned study 7. Briefly, pregnant C57B1 mice (Iffa Credo, Lyon, France) received single i.p. injections of 5/~Ci/g of [3H]thymidine (30 Ci/mMol, C . E . A . , Saclay, France) between embryonic days E l 0 and El7. Two pregnant females were injected at E l 0 , 13, 14 or 17, and one at E l l , 12, 15 or 16. Mating day, ascer-
tained by the presence of a vaginal plug, was considered as E0. Offspring were sacrificed at 3 months of age. From each litter, two animals were used in the present experiment. Under chloral hydrate anesthesia, the animals received a bilateral injection Of ]/~1 colchicine 16 in saline (20/~g//d) in the cortex overlying the dorsal hippocampus. After a survival time of 24 h they were perfused, under ether anesthesia, with p e r i o d a t e - l y s i n e - 4 % paraformaldehyde fixative 13. Coronal 20-#m-thick sections were cut with a freezing microtome and processed for G A D immunocytochemistry using the P A P method 22. The characteristics of the anti-GAD antibody (courtesy of Dr. M.L. Tappaz, Lyon, France) have been reported elsewhere 15. Triton X-100 was used to increase penetration of the immunoreagents. Sections were mounted on chrome-alum gelatinized slides and air dried. The sections were defatted, dipped in NTB-2 and exposed at 4 °C for one month before development in D19. Examples of [3H]thymidine-labeled GAD-positive cells are shown in Fig. 1. The clusters of silver grains (arrows) over unstained structures were interpreted to label n o n - G A D neurons. The possibility that the
* A preliminary report of this work has been published (ref. 21). Correspondence: E. Soriano, Departamento de Morfologia Microsc6pica, Facultad de Biologia, Universidad de Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain 0165-3806/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
Fig. 1. Illustrations of [3H]thymidine-labeled GAD-positive and non-GAD cells. Nomarski photomicrographs. Bar = 25/~m. A: two GAD-positive cells, probably pyramidal basket cells, located in the deep aspect of the granular layer (SG) of area dentata. Both were heavily labeled by [3H]thymidine after a pulse injection at El4. H, hilus. B: a multipolar GAD-positive cell located in the inferior zone of the stratum moleculare (SM) of area dentata. It became labeled after a pulse at El4. An arrow points to a cluster of silver grains labeling a non-GAD cell in stratum granulosum (SG). C: a GAD-positive cell in stratum radiatum (SR) of regio inferior, labeled by [3H]thymidine at E13. A cluster of silver grains (arrow) marks the presence of a non-GAD cell in stratum pyramidale (SP). D: two GAD-positive cells in the inferior aspect of the stratum pyramidale of regio inferior. One of them was labeled by [3H]thymidine at El3. E: [3H]thymidine-labeled cells in the inferior aspect of stratum pyramidale of regio superior after a pulse at El4. One of them is GAD-positive. Arrows point to labeled non-GAD cells.
90 clusters were associated to glial cells was ruled out (see refs. 7 and 19 for discussion). The positions of labeled cells were plotted onto camera lucida charts. Per injection day, a total of 32 such charts were made for GAD-positive cells, and 8 for n o n - G A D cells. The charts were taken from at least two littermates labeled at any given embryonic day. Animals from two different litters were used in the case of E l 3 , E l 4 and E l 7 injections. Numerical densities (number of labeled cells per m m 2) were calculated. In each major anatomical subdivison (area dentata, regio inferior and regio superiorS), the distributions of labeled GAD-positive and n o n - G A D cells (numerical densities vs. embryonic days, see Fig. 2) were analyzed using one-way analysis of variance and Tukey's multiple comparison test 24. The first [3H]thymidine-labeled n o n - G A D cells were observed after E l l injections. These labeled cells were present, in very small numbers, in all subdivisions of the hippocampus. The evolution of the labeling can be followed in Fig. 2 (dashed lines). In area dentata, the labeling of n o n - G A D cells increased steadily during the whole embryonic period studied. Most of these n o n - G A D cells were located in the granular layer and must correspond to granule cells, of which a large proportion is generated postnatally L3'4'17. On the other hand, n o n - G A D cells in the hilus, many of which correspond to modified pyramids, were labeled between E12 and El4. In the hippocampus proper, no major differences were found with previous autoradiographic analyses in the mouse 3,6. Thus, in the regio inferior, n o n - G A D cells reached a peak of labeling at E l 4 and El5. In the regio superior, the maximum labeling of n o n - G A D cells started at E l 5 and lasted until El6. A regio inferior-regio superior gradient, as defined in previous studies 3'4']8, is evident (Fig. 2). In area dentata, GAD-positive cells (Fig. 2, continuous line) reached their maximum labeling between E l 3 and E l 4 , and showed a clear decline during successive days of injection. After pulses at E l 7 , only a few GAD-positive cells became labeled. This labeling pattern is compatible with the idea that GAD-positive cells in area dentata are generated prenatally 2,~°. In the hippocampus proper, maximum labeling of GAD-positive cells started earlier than in area dentata. In regio inferior, this maximum was reached al-
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Fig. 2. Mean numerical densities (number of cells per mm2) and S.E.M.s of radioactively labeled cells at Ell-E17. Non-GAD cells, dashed lines; GAD-positive cells, continuous lines. Data were obtained from camera lucida charts. Area dentata: the labeling of non-GAD cells increases steadily during the embryonic period analyzed. A maximum of GAD-positive cell labeling occurs at El3 and El4. Differences exist between El4 and El2, and between El4 and El5 (P < 0.01). Regio inferior: the maximum of non-GAD cell labeling occurs at El4 and El5 (P < 0.01). GAD-positive cells reach their maximum labeling at El2 and El3 (P < 0.01). Regio superior: the maximum labeling of non-GAD cells is observed after injections at El5 and El6. Significant differences are found between El5 and El4 (P < 0.05) and between El6 and El7 (P < 0.01). The highest GAD-positive cell labeling is found between El2 and El5. Differences exist between El2 and Ell (P < 0.01) and between El5 and El6 (P < 0.05).
ready at E12 and E l 3 but decreased abruptly thereafter, reaching zero levels at El6. In regio superior, the maximum labeling occurred from E l 2 through
91
Fig. 3. Camera lucida drawings of the dorsal hippocampus showing the locations of GAD-positive cells labeled by [3H]thymidine at E12-E16. The cells found in 4 adjacent sections were pooled together onto each single drawing. See text for details. RI, regio inferior; RS, regio superior; AD, area dentata.
El5, and very few GAD-positive cells became labeled at E16 and E17. Similarly to GAD-negative cells, GAD-positive cells followed a regio inferiorregio superior gradient of neurogenesis. We conclude that, in the hippocampus proper, the peaks of neurogenesis occur earlier for the GAD-positive than for the GAD-negative cells. In both hippocampal subdivisions, but especially in the regio superior, GAD-positive cells showed a distinctive radial gradient (sandwich gradient), illus-
trated in Fig. 3. Injections at E l 2 resulted in the labeling of GAD-positive cells in the stratum oriens (close to the alveus), in the upper part of the stratum radiatum, and in the interface between the latter and the stratum lacunosum-moleculare. Injections at El3 resulted in a widespread distribution of labeled neurons among all layers of the hippocampus, including the stratum pyramidale. Injections from E l 4 onwards labeled GAD-positive cells in the stratum pyramidale and in the adjacent parts of the stratum radiatum and stratum oriens. This sandwich gradient has been detected previously in conventional autoradiographic studies 4'8, but the present results suggest that it is mainly related to the differentiation of GAD-positive cells. A detailed account on neurogenetic gradients of GAD-positive cells in the mouse hippocampus is in preparation. It is generally accepted that most hippocampal GABAergic neurons are interneurons 16, although there may be some exceptions z°. The vast majority of the non-GABAergic neurons (pyramidal and granule cells) are projection neurons. Thus, and contrarily to the generally accepted time sequence of neurogenesis 9, in the hippocampus the local circuit neurons are born earlier than the projection neurons. By combining [3H]thymidine autoradiography and immunocytochemistry, we were able to visualize the particular spatiotemporal gradients of generation of the GAD-immunoreactive neuron population which otherwise could be masked by the major neuronal types of the same region. The reasons for the differences with neocortical neurogenesis 7'14 are still unclear. It is in the marginal and intermediate zones of the developing neocortex where the first postmitotic cells can be recognized 11"12, and where the earliest GABAergic cells are identifiable 23. Differences become less conspicuous if we compare the adult hippocampus with the postnatal neocortex, before massive cell death reported for the marginal and intermediate zones of neocortex la has occurred. We thank C. Sotelo and M. Wassef for their advice, M.L. Tappaz for providing the anti-GAD antibody, and J.P. Rio, F. Roger and J. Simmons for technical help. Supported by the Fondation pour la Recherche M6dicale (France), CNRS (France) and CSIC/CAICYT (Spain), Grant 154/1985.
92 1 Altman, J. and Das, G., Autoradiographic and histological evidence of postnatal neurogenesis in rats, J. Comp. NeuroI., 124 (1965) 319-336. 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., 59 (1985) 33-39. 3 Angevine, J.B., Jr., Time of neuron origin in the hippocampal region: an autoradiographic study in the mouse, Exp. Neurol., Suppl. 13 (1965) 1-70. 4 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. 5 Blackstad, T.W., Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination, J. Comp. Neurol., 105 (1956) 417-538. 6 Caviness, V.S., Jr., Time of neuron origin of the hippocampus and dentate gyrus of normal and reeler mutant mice: an autoradJographic analysis, J. Comp. Neurol., 151 (1973) 113-120. 7 Fair6n, A., Cobas, A. and Fonseca, M., Times of generation of glutamic acid decarboxylase immunoreactive neurons in mouse somatosensory cortex, J. Comp. Neurol., in press. 8 Hine, R.J. and Das, G.D., Neuroembryogenesis in the hippocampal formation of the rat. An autoradiographic study Z. Anat. Entwickl.-Gesch., 144 (1974) 173-186. 9 Jacobson, M., Developmental Neurobiology, Plenum, New York, 1978, 562 pp.. 10 LUbbers, K., Wolff, J.R. and Frotscher, M., Neurogenesis of GABAergic neurons in the rat dentate gyrus: a combined autoradiographic and immunocytochemical study, Neurosci. Lett., 62 (1985) 317-322. 11 Luskin, M.B. and Shatz, C.J., Studies of the earliest generated cells of the cat's visual cortex: cogeneration of subplate and marginal zones, J. Neurosci., 5 (1985) 1062-1075. 12 Marfn-Padilla, M., Early prenatal ontogenesis of the cerebral cortex (neocortex) of the cat (Felis domestica). A Golgi study. I. The primordial neocortical organization, Z. Anat. Entwickl.-Gesch., 134 (1971) 117-145. 13 McLean, I.W. and Nakane, P.K., Periodate lysine-paraformaldehyde fixative. A new fixative for immuno-electron
14
15
16
17
18
19
20
21
22
23
24
microscopy, J. Histochem. Cytochem., 22 (1974) 1077-1083. Miller, M.W., Cogeneration of retrogradely labeled corticocortical projection and GABA-immunoreactive local circuit neurons in cerebral cortex. Dev. Brain Res., 23 (1985) 187-192. Oertel, W.H., Schmechel, D.E., Tappaz, M.L. and Kopin, I.J., Production of a specific antiserum to cat brain glutamic acid decarboxylase by injection of an antigen-antibody complex, Neuroscience. 6 (1981) 2689-2700. Ribak, C.E., Vaughn, J.E. and Saito, K., Immunocytochemical localization of glutamic acid decarboxytase in neuronal somata following colchicine inhibition of axonal transport, Brain Res., 140 (1978) 315-332. Schlessinger, A.R., Cowan, W.M. and Gottlieb, D.I., An autoradiographic study of the time of origin and the pattern of granule cell migration in the dentate gyrus of the rat, J. Comp. Neurol., 159 (1975) 149-176. Schlessinger, A.R., Cowan, W.M. and Swanson, L.W., The time of origin of neurons in Ammon's horn and the associated retrohippocampal fields, Anat. Embryol., 154 (1978) 153-173. Schmechel, D.E. and Rakic, P., A Golgi study of radial glial cells in developing monkey telencephalon: morphogenesis and transformation into astrocytes, Anat. Ernbrvol., 156 (1979) 115-152. Seress, L. and Ribak, C.E., GABAergic cells in the dentate gyrus appear to be local circuit and projection neurons, Exp. Brain Res., 50 (1983) 173-182. Soriano, E., Cobas. A., Tappaz, M.L. and Fair6n. A., Times of origin of GAD positive neurons in the mouse hippocampus and area dentata, Neurosci. Lett., Suppl. 22 (1985) $330 (Abstract).. Sternberger, L.A., Hardy, P.H., Cuculis, J.J. and Meyer, H.G., The unlabeled antibody method of immunohistochemistry. Preparation and properties of soluble antigen complex, and its use in identification of spirochetes, J. Histochem. Cytochem., 18 (1970) 315-333. Wolff, J.R., Bottcher, H., Zetzsche. T., Oertel, W.H. and Chronwall, B.M., Development of GABAergic neurons it, rat visual cortex as identified by glutamate decarboxylaselike immunoreactivity, Neurosci. Lett., 47 (1984) 207-212. Zar, J.H., Biostatistical analysis, Prentice-Hall, Englewood Cliffs, 1984, pp. 163,186.