Transient calretinin expression de nes early postmitotic step of
neuronal differentiation in adult hippocampal neurogenesis of mice
Moritz D. Brandt,
a,1
Sebastian Jessberger,
b,1
Barbara Steiner,
b
Golo Kronenberg,
a,c
Katja Reuter,
a
Anika Bick-Sander,
b
Wolfger von der Behrens,
a
and Gerd Kempermann
a,b,
*
a
Max Delbru¤ck Center for Molecular Medicine (MDC) Berlin-Buch, Robert-Ro¤ssle-Strasse 10, 13125 Berlin, Germany
b
VolkswagenStiftung Research Group, Department of Experimental Neurology, Charite· University Hospital, Humboldt University,
Schumannstrasse 20/21, 10117 Berlin, Germany
c
Department of Psychiatry, Freie Universita¤t Berlin, Eschenallee 3, 14050 Berlin, Germany
Received 10 April 2003; revised 4 June 2003; accepted 6 June 2003
Abstract
We here show that the early postmitotic stage of granule cell development during adult hippocampal neurogenesis is characterized by
the transient expression of calretinin (CR). CR expression was detected as early as 1 day after labeling dividing cells with bromodeoxyuri-
dine (BrdU), but not before. Staining for Ki-67 con rmed that no CR-expressing cells were in cell cycle. Early after BrdU, CR colocalized
with immature neuronal marker doublecortin; and later with persisting neuronal marker NeuN. BrdU/CR-labeled cells were negative for
GABA and GABAA1 receptor, but early on expressed granule cell marker Prox-1. After 6 weeks, no new neurons expressed CR, but all
contained calbindin. Stimuli inducing adult neurogenesis have limited (enriched environment), strong (voluntary wheel running), and very
strong effects on cell proliferation (kainate-induced seizures). In these models the induction of cell proliferation was paralleled by an
increase of CR-positive cells, indicating the stimulus-dependent progression from cell division to a postmitotic stage.
' 2003 Elsevier Inc. All rights reserved.
Introduction
In most studies to date, adult hippocampal neurogenesis
has been investigated by assessing two key events: the
proliferation of the progenitor cells in the dentate gyrus and
the survival of the progeny and expression of neuronal
markers about 4 weeks later (Kuhn et al., 1996). Much less
is known about the period of neuronal development in
between. It appears, however, that the fate choice decision
of new neurons is made early and that those cells that
express neuronal markers such as NeuN are likely to be-
come persistently integrated into the granule cell layer
(Kempermann et al., 2003). A much larger proportion of
newborn cells express immature neuronal markers such as
doublecortin (DCX) or
-III-tubulin, and it is suggestive
that this group of cells represents a pool of potential new
neurons, from which under conditions that stimulate adult
neurogenesis more new cells can be recruited for neuronal
development (Kempermann et al., 1997b; Gould et al.,
1999). At least a subset of DCX-expressing cells is highly
proliferative (Filippov et al., 2003), and it is reasonable to
assume that this group of cells contains lineage-determined
neuronal progenitor cells. How can the developmental
point-of-no-return be de ned, at which these cells become
postmitotic and enter de nite neuronal development?
This question is important for a number of reasons: (1)
precision about the time point when cells become postmi-
totic will allow the fate choice decision in adult hippocam-
pal neurogenesis to be pinpointed; (2) better characteriza-
tion of early postmitotic neuronal development will allow
identi cations of the factors within the cells and in their
local environment that control this process; (3) compari-
sions as early adult neurogenesis with net adult neurogen-
esis quantitatively will allow dissection of the effects of
* Corresponding author. Max Delbru‹ck Center for Molecular Medicine
(MDC), Berlin-Buch, Robert-Ro‹ssle-Str. 10, 13125 Berlin, Germany. Fax:

49-30-9406-3814.
E-mail address: gerd.kempermann@mdc-berlin.de (G. Kempermann).
1
Contributed equally to this work.
Molecular and Cellular Neuroscience 24 (2003) 603—613 www.elsevier.com/locate/ymcne
1044-7431/03/$ — see front matter ' 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S1044-7431(03)00207-0

manipulations aiming at stimulating neurogenesis; (4) char-
acterizations of adult neuronal development in detail and
over its time course will lead to a clearer picture of how the
new neurons grow into their functional connections and thus
how they might contribute to hippocampal function; and
nally (5) identi cations of vulnerable development steps in
cases of disturbed adult hippocampal neurogenesis, as it is
for example discussed for epilepsy (Parent and Lowenstein,
2002) or major depression (Jacobs et al., 2000), will help in
understanding how failing adult neurogenesis might con-
tribute to disease.
A chance observation sparked the series of experiments
we report here. Incidentally we found that in the adult
murine subgranular zone (SGZ) a number of cells expressed
calretinin (CR) and contained proliferation marker bro-
modeoxyuridine (BrdU) that had been injected into the
animals 4 weeks earlier. As we will see in the following
paragraphs, our initial hypothesis that these cells might be
newly generated interneurons turned out to be wrong. We
therefore set out to study the role of calretinin-expressing
cells within the context of adult hippocampal neurogenesis.
It soon became evident that CR expression was linked to
early neuronal development in adult hippocampal neurogen-
esis. A report by Liu et al., (1996) has speculated that
CR-containing cells might be immature granule cells, but so
far, this idea has not been proven. The rationale of the study
presented here therefore was to characterize calretinin-ex-
pressing cells qualitatively and quantitatively within the
context of adult hippocampal neurogenesis and relate cal-
retinin expression to identi able milestones of neuronal
development.
Results
Newly generated cells in the adult SGZ express calretinin
Mature granule cells in the adult dentate gyrus are known
to express calbindin (Sloviter, 1989) and many subpopula-
tions of interneurons can be categorized by their expression
of other calcium-binding proteins such as parvalbumin or
CR (Freund and Buzsaki, 1996). In order to nd out whether
newly generated neurons in the adult dentate gyrus were
heterogeneous with regard to their expression of calcium-
binding proteins, we examined BrdU-labeled, newly gener-
ated cells for their expression of parvalbumin and CR.
Nowhere in the SGZ were BrdU-labeled parvalbumin-ex-
pressing cells detected (Fig. 1A). However, at 4 weeks after
BrdU approximately 6% of the BrdU-labeled cells ex-
pressed CR (Figs. 1B and 2A). We have reported earlier that
at 4 weeks after BrdU, about 60 to 70% of the BrdU-labeled
cells express calbindin (Kempermann et al., 1997a).
CR-positive cells were localized in the SGZ of the gran-
ule cell layer. There were no distinguishable differences in
the localization of CR- or calbindin-expressing newborn
cells, and the expression of both CR and calbindin in BrdU-
positive cells colocalized with neuronal marker NeuN (Fig.
1B). At this time point, in the SGZ the overlap was 100%:
there were no CR-positive cells that were NeuN-negative. In
order to allow extensive combinations of primary antibodies
from different species, three different anti-CR-antibodies
(from mouse, goat, and rabbit) were used in the present
study: They all yielded identical results.
Thus, with regard to the expression of calretinin or cal-
bindin two populations of newly generated neurons could be
identi ed at 4 weeks after BrdU. The question was whether
this re ected the existence of two stable independent pop-
ulations or snapshots from a dynamic developmental pro-
cess.
Calretinin is transiently expressed in maturing new
neurons
In order to assess the kinetics of CR expression in newly
generated cells we determined at which time point CR was
rst expressed in newly generated neurons. For these time
course studies animals were examined at 4 h, 1 day, 3 days,
7 days, 2.5 weeks, and 4 weeks after a single injection of
BrdU.
CR expression in newborn cells was observed as early as
1 day after BrdU, reaching a maximum after one week (76.6
 1.1%; Fig. 2A). Thereafter CR-expressing BrdU-positive
cells decreased in number. In absolute terms, the number of
BrdU/CR double-labeled cells peaked at 3 days after BrdU
and decreased over the following weeks (Fig. 2B). At 6
weeks after BrdU no BrdU/CR double-labeled cells could
be detected (not shown). This nding suggested that CR is
expressed transiently in BrdU-labeled cells.
Doublecortin is a key molecule in cortical and hippocam-
pal development (des Portes et al., 1998; Corbo et al., 2002)
and is expressed transiently in adult hippocampal neurogen-
esis (Kempermann et al., 2003). At 1 day after BrdU we
found a large percentage of BrdU-positive cells that con-
comitantly showed CR and DCX immunoreactivity (Fig.
1B). This overlap was not found at 4 weeks after BrdU (Fig.
1B).
As evident from the time course in Fig. 2B the increase
in the absolute number of newborn CR-positive cells par-
alleled the increase in the number of BrdU-marked NeuN-
positive cells. The number of BrdU/NeuN-double-labeled
cells, however, remained stable after 2.5 weeks, whereas
BrdU/CR-double-labeled cells faded out. From the nding
that all CR-positive cells expressed NeuN and the overlap
between CR and DCX at early time points, we concluded
that CR is transiently expressed in newly generated neurons
at early stages of their development.
Newly generated CR-positive cells do not express GABA,
GABAA1 receptor, or reelin
The nding that CR is transiently expressed in immature
neurons of the adult dentate gyrus did not allow determina-
604 M.D. Brandt et al. / Molecular and Cellular Neuroscience 24 (2003) 603 613