Depletion of central BDNF in mice impedes terminal differentiation of new granule neurons in the adult hippocampus

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Abstract

Granule neurons generated in the adult mammalian hippocampus synaptically integrate to facilitate cognitive function and antidepressant efficacy. Here, we investigated the role of BDNF in facilitating their maturation in vivo. We found that depletion of central BDNF in mice elicited an increase in hippocampal cell proliferation without affecting cell survival or fate specification. However, new mutant neurons failed to fully mature as indicated by their lack of calbindin, reduced dendritic differentiation and an accumulation of calretinin+ immature neurons in the BDNF mutant dentate gyrus. Furthermore, the facilitating effects of GABAA receptor stimulation on neurogenesis were absent in the mutants, suggesting that defects might be due to alterations in GABA signaling. Transcriptional analysis of the mutant hippocampal neurogenic region revealed increases in markers for immature neurons and decreases in neuronal differentiation facilitators. These findings demonstrate that BDNF is required for the terminal differentiation of new neurons in the adult hippocampus.

Introduction

The hippocampus is a site of active neurogenesis throughout life in mammals, including humans (Kempermann et al., 1997a, Kempermann et al., 1997b, Eriksson et al., 1998, Gould et al., 1999). New granule neurons generated through this form of brain plasticity functionally integrate into the dentate gyrus to facilitate cognitive function (van Praag et al., 2002, Ramirez-Amaya et al., 2006). Indeed, interfering with this process hinders some forms of hippocampal-dependent learning, long-term potentiation and antidepressant efficacy (Santarelli et al., 2003, Snyder et al., 2005, Saxe et al., 2006). Adult neurogenesis is dynamically regulated by environmental manipulations, neurotransmitters and endocrine and growth factors (Warner-Schmidt and Duman, 2006). However, the signaling mechanisms that influence the proliferation, survival and differentiation of progenitors in this brain area have not been fully elucidated. Because of their established roles in the developing nervous system, brain-derived neurotrophic factor (BDNF) and its receptor TrkB quickly emerged as candidate modulators of adult neurogenesis. However, the intricacies of their actions remain unknown.

BDNF appears to contribute to the neurogenic milieu that supports precursors in the adult hippocampus. Evidentiary is the finding that BDNF+/āˆ’ mutant mice exhibited reduced survival of new cells in this region (Sairanen et al., 2005). Furthermore, BDNF haploinsufficiency suppressed the enhancing effects of dietary restriction and antidepressant treatment on hippocampal neurogenesis (Lee et al., 2002, Santarelli et al., 2003). The subgranular zone (SGZ) of the dentate gyrus contains two distinct populations of cycling progenitors (type 1 and 2 cells) and newly post-mitotic cells in different stages of differentiation (Ambrogini et al., 2004, Garcia et al., 2004). Type 1 precursors are putative stem cells that express glial fibrillary acidic acid (GFAP) and give rise to type 2 progenitors, which are neuronal lineage-restricted precursors and contain GABAA receptors (Seri et al., 2001, Fukuda et al., 2003, Garcia et al., 2004). Because of their high intracellular levels of chloride, GABA exerts a depolarizing effect on these cells, promoting their exit from the cell cycle and differentiation into granule neurons (LoTurco et al., 1995, Wang et al., 2000, Ge et al., 2005, Tozuka et al., 2005, Wang et al., 2005).

It remains unclear at which stages BDNF might act to modulate development of neural precursors in the adult hippocampus and whether its actions influence cell fate specification and terminal differentiation of these cells in vivo. It was recently reported that TrkB expression in adult-generated hippocampal neurons increases as these cells develop (Donovan et al., 2008). As BDNF mediates neuronal differentiation during early development (Patapoutian and Reichardt, 2001), it is plausible that it acts on newly post-mitotic cells to direct their maturation into functional granule neurons in the adult hippocampus. Moreover, because this neurotrophin plays a critical regulatory role in GABAergic signaling in the hippocampus and other brain areas (Brunig et al., 2001, Elmariah et al., 2004, Henneberger et al., 2005), its actions could involve facilitating the effects of GABA. To test this, we examined adult hippocampal neurogenesis in mutant mice with depletion of central BDNF. We found that they exhibited deficits in the terminal differentiation, dendritic complexity and migration of new granule neurons and blunted GABAA-mediated effects on neurogenesis. These findings demonstrate that BDNF is an essential constituent of the neurogenic microenvironment in the adult hippocampus.

Section snippets

Proliferating and post-mitotic neuronal precursors in the adult hippocampus contain TrkB receptors

Little is known regarding the effects of BDNF on actively dividing cells in the adult dentate gyrus or on new cells that recently exited the cell cycle to follow a neuronal differentiation pathway. To gain insight into the actions of BDNF, we examined TrkB expression in cells at different developmental stages in the adult hippocampus as an indicator of their ability to respond to this neurotrophin. To distinguish proliferating cells in wild type mice, we administered BrdU, a thymidine analog,

Discussion

BDNF has been suspected to play an influential role in adult hippocampal neurogenesis. Evidentiary are reports indicating altered cell proliferation and diminished enhancement of neurogenesis by antidepressant treatment or dietary restriction in the hippocampi of BDNF+/āˆ’ mice (Lee et al., 2002, Sairanen et al., 2005). However, the specific effects of BDNF in the differentiation of adult-born granule neurons in vivo remained largely unknown. Here, we show that BDNF is required for later phases

Animals

Mice with postnatal depletion of central BDNF (BDNF2L/2LCk-cre) were generated as described previously (Rios et al., 2001). Briefly, for the generation of mice with floxed BDNF alleles, loxP sites were inserted around the single coding exon of BDNF. Thus, cre-mediated recombination of floxed BDNF results in a null BDNF allele. BDNF conditional mutants were generated by crossing mice carrying floxed BDNF alleles with transgenic mice in which expression of cre recombinase was driven by the

Acknowledgments

The authors would like to thank Leila Bradley for review of the manuscript, Sevin Turcan for assistance with the interpretation of the microarray data and the Imaging and Genomics core facilities at Tufts University for facilitating these studies. These cores are supported by the Center for Neuroscience Research (P30 NS047243) and GRASP (P30 DK34928). This work was supported by an Alfred P. Sloan Foundation fellowship and NIH/NIMH grant R01 MH67817 to M.R.

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