Conditional labeling of newborn granule cells to visualize their
integration into established circuits in hippocampal slice cultures
and Beat H. Ga¨hwiler
Brain Research Institute, University of Zurich, Zurich, Switzerland
Biozentrum, University of Basel, Basel, Switzerland
Friedrich Miescher Institute, Basel, Switzerland
Institut de Neurosciences cellulaires et integratives, Strasbourg, France
Brain Repair Centre, University of Cambridge, UK
Received 8 February 2006; revised 9 May 2006; accepted 19 May 2006
Available online 7 July 2006
The dentate gyrus continues to produce new granule cells throughout
life. Understanding the mechanisms underlying their integration into
the pre-existing hippocampal circuitry is of crucial importance. In the
present study, we developed an approach allowing visual tracking of
newborn granule cells in hippocampal organotypic slices. By crossing
neurogenin 2 (Ngn2-CreERi) with Cre-reporter mice expressing YFP
or GFP reporter genes, it was possible to observe living cells after
treating slice cultures with 4-hydroxytamoxifen to induce Cre recom-
binase activation. Colocalization of GFP with the mitotic marker BrdU
demonstrated that the GFP-expressing granule cells were born in vitro.
They mature and integrate normally into the hippocampal circuitry, as
shown using morphological and electrophysiological techniques. This
ex vivo approach therefore offers a highly accessible model to study the
effects of long-term treatments on maturation and integration of
newborn granule cells.
D 2006 Elsevier Inc. All rights reserved.
The persistence of neurogenesis in the dentate gyrus throughout
life is a characteristic feature of the hippocampus, a structure
involved in learning and memory formation. Neuronal progenitors
that proliferate in the subgranular zone give rise to new granule
cells that successfully integrate following a series of highly
stereotyped maturation steps (Ge et al., 2006; van Praag et al.,
2002; Esposito et al., 2005; Zhao et al., 2006). These newly
generated granule cells receive functional synaptic inputs that
exhibit enhanced synaptic plasticity (van Praag et al., 2002;
Schmidt-Hieber et al., 2004). An increasing number of studies
indicates a link between granule cell neurogenesis, memory
formation and cognition (McEwen, 1999; van Praag et al., 1999;
Shors et al., 2001).
As important as the birth of new granule cells is their
maturation and correct integration into the pre-existing hippocam-
pal circuitry. Improper integration of newborn granule cells and
their axons may have a dramatic impact on their survival and more
generally on the function of the hippocampal formation, possibly
leading to epileptogenesis, chronic seizures and depression
(Schwartzkroin, 1994; Parent et al., 1997; Parent et al., 1999;
Parent and Lowenstein, 2002). Despite its significance, only few
studies have addressed the mechanisms underlying the integration
of newborn granule cells within the hippocampal circuitry. In part,
this is due to the problems involved in tracking newborn neurons at
the single cell level.
Here, we present a Cre-Lox approach allowing reporter gene
expression to be induced at a given time in neuronal progenitors in
organotypic slice cultures. This technique thus allows detailed
analysis of the morphology and the development of newborn
granule cells and of their integration into neuronal circuits under
well-controlled culture conditions.
Visualization of newborn granule cells in hippocampal slices
The dentate gyrus continues to produce granule cells postna-
tally well into adulthood. To investigate the integrative capability
of postnatally generated neurons and to study the factors
influencing this process, newly generated cells need to be directly
visualized. We used a conditional approach to induce fluorescent
reporter gene expression in neuronal progenitors by taking
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* Corresponding author. Current address: Cambridge Centre for Brain
Repair. E.D. Adrian Building, Forvie site, Robinson Way, CB2 2PY
E-mail address: firstname.lastname@example.org (O. Raineteau).
Available online on ScienceDirect (www.sciencedirect.com).
Mol. Cell. Neurosci. 32 (2006) 344 – 355