Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
Origin of the Neocortical Subependymal Cells Speculated by Emx1 and GAD67 Expression
Department of Morphological Neural Science, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, 860-8556 Japan
Correspondence to be sent to: Nobuaki Tamamaki, e-mail: tamamaki{at}kaiju.medic.kumamoto-u.ac.jp
Key words: GABA, GABAergic neuron, neocortex, neurogenesis, olfactory bulb, subependymal layer, subventricular zone
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Introduction |
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More than a decade has passed since adult neurogenesis in the murine telencephalon was described and accepted widely. The adult neurogenesis has been attributed to the progenitors in the subgranular layer in the dentate gyrus and the subependymal layer of the telencephalon. However, the developmental origin of the progenitors in the dentate gyrus and the subependymal layer is poorly understood. It is well known that the neurogenesis in the embryo brains occurs in the ventricular zone (VZ). Recently it has been proposed that the progenitors in the VZ of the telencephalon turn into the subventricular zone (SVZ) astrocytes (Tramontin et al., 2003
).
The subependymal layer of the neocortex has been regarded as a source of the granule
cells and periglomerular cells in the adult olfactory bulb. The subependymal layer
produces neurons that migrate to the olfactory bulb as the rostral migratory stream, and
the newly produced neurons are going to be GABAergic inhibitory neurons in the olfactory
bulb. However, we felt discontinuity in the idea that the neocortical VZ cells turn into
SVZ astrocytes and then produce GABAergic neurons. By now, it is well known that most of
the neocortical GABAergic neurons were supplied by tangential cell migration originating
in the subpallial structures (Anderson et
al., 1997;
Tamamaki et al., 1997). It is
also speculated that the neocortical VZ almost lack the ability to produce GABAergic
neurons. Under these circumstances, we speculated that the GABAergic neurons for the
olfactory bulb may be produced by progenitors derived from other than the neocortex. In
this paper we will show that the subependymal cells in the adult neocortex are
heterogeneous and may have different origin.
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Materials and methods |
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TopIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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Animals, histology and histochemistry
Mice used in this study were obtained by mating homozygous Emx1-Cre knock-in mice
(Iwasato et al., 2000) and
Cre-mediated lacZ/AP (alkaline phosphatase) double reporter mouse (Lobe et al., 1999) or by mating heterozygous
GAD67-GFP knock-in neo+ mice (Tamamaki et
al., 2003) with wild-type mice. These mice were anesthetized with
pentobarbital (50 mg/kg) and perfused at 3 months old with saline and a fixative
containing 4% formaldehyde in phosphate buffer (pH 7.4). After post fixation with
the same fixative and cryoprotection, cryostat sections (12–50 µm) were made
and processed for histochemistry or observation. For histochemistry of lacZ and AP, we
followed the methods described previously (Lobe
et al., 1999).
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Results |
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TopIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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In this study we started to investigate how the neocortical subependymal cells obtained the ability to produce GABAergic neurons in the neocortex. First, we examined whether the neocortical subependymal cells were on the Emx1-negative cell lineage or Emx1-positive cell lineage.
We mated homozygous Emx1-Cre knock-in mice and Cre-mediated lacZ/AP double reporter
mouse (Lobe et al., 1999). In
these mice, cells in Emx1-negative cell lineage appear as positive for lacZ and cells in
Emx1-positive cell lineage appear as positive for AP. We investigated the subependymal
layer in this mouse after X-gal reaction for lacZ and NBT reaction for AP (Figure
1A). Vast majority of the neocortical
neurons were positive for AP and revealed as Emx1-positive cell lineage. The majority of
the subependymal cells were also positive for AP as a continuation of the neocortex.
However, the AP-positive cell layer was interrupted by lacZ-positive cell clusters in the
subependyma (Figure
1A). Several lacZ-positive cell
clusters were found in every frontal section at 50 µm thickness. The size of the
cell cluster varied much and we counted the lacZ-positive cells in a cluster from a few
to close to a hundred.
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We also investigated the characteristics of cells in the subependymal layer of the GAD67-GFP knock-in mouse (Tamamaki et al., 2003
). We found several GFP-positive cell clusters in every frontal
section at 50 µm thickness (Figure
1B). The number of GFP-positive cells
in a cluster ranged from a few to nearly a hundred.
We also found the lack of Emx1 immunoreactivity in the GAD67-GFP-positive cells in
the knock-in mouse neocortex and reported it previously (Nakamura et al., 2003). Thus the lacZ-positive cell
clusters seemed to correspond to the GAD67-GFP-positive cell clusters in the subependymal
layer.
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Discussion |
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TopIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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We used the Emx1-Cre knock-in mouse and the GAD67-GFP knock-in mouse to reveal heterogeneity in the subependymal cells in the adult mouse neocortex. It has been well documented that Cre expression in the former mouse and GFP expression in the latter mouse were regulated by Emx1 promoter and GAD67 promoter properly (Iwasato et al., 2000
;
Tamamaki et al., 2003). Thus,
we interpreted the AP reporter expression as Emx1 expression and GFP expression as GAD67
expression in the neocortex.
According to the distribution, size and number of the lacZ-positive cell clusters and
the GAD67-GFP-positive cell clusters, they seemed like a structure almost completely
overlapping each other. In good agreement, the GAD67-GFP-positive cells lacked
Emx1-immunoreactivity in the knock-in mouse (Nakamura et al., 2003). Since Emx1 has been
regarded as primarily a pallial marker, it was discussed repeatedly that neocortical
cells which lack the Emx1 expression might be derived from the subpallial structures
(Chan et al., 2001;
Gorski et al., 2002). Thus we
speculate that the Emx1-negative cells in the subependymal layer are derived from the
subpallial structures and the Emx1-positive cells are derived from the pallium. Although
our speculation depends exclusively on whether the Emx1 is really a reliable pallial
marker, we believe that Emx1 expression indicates that the subependymal cells in the
neocortex are heterogeneous. In the case of Emx1-negative subependymal cells
proliferating and functioning in adult neurogenesis, the major products of them will be
inhibitory GABAergic neurons.
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Summary |
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TopIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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The subependymal layer of the telencephalon has been regarded as a source of the granule cells and periglomerular cells in the adult olfactory bulb. However, the developmental origin of the neocortical subependymal layer is poorly understood. Here we examined the expression of a neocortical marker, Emx1, in the adult neocortical subependymal cells in mice. The subependymal cells were heterogeneous, some of them being of an Emx1-negative cell lineage and others being Emx1-positive. The Emx1-negative cell clusters seemed to overlap with GAD67-postive cell clusters in the subependymal layer. These observations imply that some of the neocortical subependymal cells were derived from the subpallial structures. When these two types of subependymal cells proliferate and function for adult neurogenesis, their product might be different.
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References |
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TopIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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Anderson, S.A., Eisenstat, D.D., Shi, L. and Rubenstein, J.L.R. (1997) Interneuron migration from the basal forebrain to the neocortex: dependence on Dlx genes. Science, 278, 474–476.
Chan, C.H., Godinho, L.N., Thomaidou, D., Tan, S.S., Gulisano, M. and Parnavelas, J.G. (2001) Emx1 is a marker for pyramidal neurons of the cerebral cortex. Cereb. Cortex, 11, 1191–1198.
Gorski, J.A., Talley, T., Qiu, M., Puelles, L., Rubenstein, J.L. and Jones, K.R. (2002) Cortical excitatory neurons and glia, but not GABAergic neurons, are produced in the Emx1-expressing lineage. J. Neurosci., 22, 6309–6314.
Iwasato, T., Datwani, A., Wolf, A.M., Nishiyama, H., Taguchi, Y., Tonegawa, S., Knopfel, T., Erzurumlu, R.S. and Itohara, S. (2000) Cortex-restricted disruption of NMDAR1 impairs neuronal patterns in the barrel cortex. Nature, 406, 726–731.[CrossRef][Medline]
Lobe, C.G., Koop, K.E., Kreppner, W., Lomeli, H., Gertsenstein, M. and Nagy, A. (1999) Z/AP, a double reporter for cre-mediated recombination. Dev. Biol., 208, 281–292.[CrossRef][Web of Science][Medline]
Nakamura, K., Nakamura, K., Kometani, K., Yanagawa, Y., Iwasato, T., Obata, K., Minato, N., Kaneko, T. and Tamamaki, N. (2003) Immigration of the proliferative progenitors for GABAergic neurons from the ganglionic eminence to the neocortex. Soc. Neurosci. Abstr., 33, 565.5.
Tamamaki, N., Fujimori, K. and Takauji, R. (1997) Origin and route of tangentially migrating neurons in the developing neocortical intermediate zone. J. Neurosci., 17, 8313–8323.
Tamamaki, N., Yanagawa, Y., Tomioka, Y., Miyazaki, J., Obata, K. and Kaneko, T. (2003) Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouse. J. Comp. Neurol., 467, 60–79.[CrossRef][Web of Science][Medline]
Tramontin, A.D., Garcia-Verdugo, J. M., Lim, D.A. and Alvarez-Buylla, A. (2003) Postnatal development of radial glia and the ventricular zone (VZ): a continuum of the neural stem cell compartment. Cereb. Cortex, 13, 580–587.
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