Pulse labeling and long-term tracing of newborn neurons in the adult subgranular zone

Abstract

Research over the past decades has demonstrated that adult brain produces neural progenitor cells which proliferate and differentiate to newborn neurons that integrate into the existing circuit. However, detailed differentiation processes and underlying mechanisms of newly generated neurons are largely unknown due to the limitation of available methods for labeling and manipulating neural progenitor cells and newborn neurons. In this study, we designed a tightly controlled, noninvasive system based on Cre/loxP recombination to achieve long-term tracing and genetic manipulation of adult neurons in vivo. In this system, tamoxifen-inducible recombinase, CreER T2 , was driven by BAC-based promoter of doublecortin (DCX, a marker of newborn neurons). By crossing this Cre line with reporter mouse, we found that newborn neurons in the dentate gyrus (DG) could be selectively pulse-labeled by tamoxifeninduced expression of yellow fluorescent protein (YFP). YFP-positive neurons were identified by coimmunostaining with cell type-specific markers and characterized by electrophysiological recording. Furthermore, analysis of the migration of these neurons showed that the majority of these labeled neurons migrated to the inner part of granule cell layer. Moreover, spine growth of inner molecular layer of newborn granule neurons takes a dynamic pattern of invert U-shape, in contrast to the wedge-shaped change in the outer molecular layer. Our transgenic tool provides an efficient way to selectively label and manipulate newborn neuron in adult mouse DG.

Genetically targeting new neurons in the adult hippocampus

“…Importantly, the labeling is permanent once the recombination occurs. By crossing the DCX-CreER mice with reporter mice for inducible expression of yellow fluorescent protein, the authors provided evidence that a majority of labeled neurons are born within a 10 day window before induction [6]. Thus, a cohort of immature neurons with relatively defined birthdates can be selectively pulse-labeled for long-term tracing and for analysis of their morphological and electrophysiological properties in the adult dentate gyrus [6].…”

“…By crossing the DCX-CreER mice with reporter mice for inducible expression of yellow fluorescent protein, the authors provided evidence that a majority of labeled neurons are born within a 10 day window before induction [6]. Thus, a cohort of immature neurons with relatively defined birthdates can be selectively pulse-labeled for long-term tracing and for analysis of their morphological and electrophysiological properties in the adult dentate gyrus [6]. Intriguingly, DCX expressing neuroblasts in the subventricular zone, the largest germinal region in the adult brain which gives rise to newborn interneurons in the olfactory bulb, were not labeled after tamoxifen induction in these mice.…”

“…The rapid progress made in understanding adult neurogenesis has been driven by new analytical tools introduced to the field, including labeling of proliferating progenitors using nucleotide analogs, such as Bromodeoxyuridine (BrdU), or 5-ethynyl-2'-deoxyuridine (EdU), and oncoretroviruses for quantitative analysis of cell proliferation, fate mapping and neuronal development [5]. In this issue of Cell Research, Xiong and colleagues bring a significant new addition to our tool box with a genetic approach, based on inducible Cre/loxP recombinase, for pulse labeling, longterm tracing and genetic manipulation of newborn neurons in the dentate gyrus of adult mice in vivo [6].…”

“…Xiong and colleagues developed a transgenic mouse line that expresses the tamoxifen-inducible recombinant CreER T2 under the control of the BAC-based promoter of doublecortin (DCX; referred to as DCX-CreER mice thereafter) [6]. DCX, a microtubuleassociated protein important for neu-npg ronal migration, is specifically and transiently expressed in proliferating neuroblasts and newborn immature neurons in both developing and adult brain [9].…”

Neuroscience in China 2010–2011

“…Recently, evidence from adult monkey and human brains suggests that neuroblasts may be generated in the subventricular zone (SVZ) and then continuously migrate to the rostral migratory stream (RMS) [2]. Furthermore, Cheng et al [3] found that newborn neurons in the dentate gyrus (DG) migrated to the inner part of the granule cell layer using a mouse model based on the Cre/loxp system, providing an efficient way to selectively label and manipulate newborn neurons in the adult mouse DG. However, the integration of newborn neurons into the existing circuit is difficult because of their poor survival, insufficient proliferation and differentiation abilities.…”