Sustained expression of brain-derived neurotrophic factor is required for maintenance of dendritic spines and normal behavior

Abstract

Brain-derived neurotrophic factor (BDNF) plays important roles in the development, maintenance, and plasticity of the mammalian forebrain. These functions include regulation of neuronal maturation and survival, axonal and dendritic arborization, synaptic efficacy, and modulation of complex behaviors including depression and spatial learning. Although analysis of mutant mice has helped establish essential developmental functions for BDNF, its requirement in the adult is less well documented. We have studied late-onset forebrain-specific BDNF knockout (CaMK-BDNF^KO) mice, in which BDNF is lost primarily from the cortex and hippocampus in early adulthood, well after BDNF expression has begun in these structures. We found that although CaMK-BDNF^KO mice grew at a normal rate and can survive more than a year, they had smaller brains than wild-type siblings. The CaMK-BDNF^KO mice had generally normal behavior in tests for ataxia and anxiety, but displayed reduced spatial learning ability in the Morris water task and increased depression in the Porsolt swim test. These behavioral deficits were very similar to those we previously described in an early-onset forebrain-specific BDNF knockout. To identify an anatomical correlate of the abnormal behavior, we quantified dendritic spines in cortical neurons. The spine density of CaMK-BDNF^KO mice was normal at P35, but by P84, there was a 30% reduction in spine density. The strong similarities we find between early- and late-onset BDNF knockouts suggest that BDNF signaling is required continuously in the CNS for the maintenance of some forebrain circuitry also affected by developmental BDNF depletion.

Introduction

BDNF, a member of the neurotrophin family of secreted proteins, was initially identified based upon its ability to support the survival of peripheral sensory neurons (Barde et al., 1982). Subsequent studies demonstrated the widespread expression of BDNF within the central nervous system (CNS) (Leibrock et al., 1989, Maisonpierre et al., 1990, Hofer et al., 1990), and its ability to influence the survival of many neuron types. BDNF has been shown to affect morphological features of specific neurons, such as soma size and dendritic arborization, as well as biochemical characteristics such as expression of biosynthetic enzymes and transporters for neurotransmitters (McAllister et al., 1999, Bibel, 2000). In addition, BDNF has been demonstrated to play a role in modulating synaptic transmission in many circuits within the brain, often potentiating synaptic strength, but sometimes reducing it (Tao and Poo, 2001, Lu et al., 2008, Waterhouse and Xu, 2009, Cunha et al., 2010, Yoshii and Constantine Paton, 2010). Given that neural activity can regulate BDNF expression and release, the fact that BDNF modulates synaptic transmission provides a molecular mechanism by which neural activity can modify the functional and structural features of neural circuitry (Thoenen, 1995, Cohen and Greenberg, 2008, Kuczewski et al., 2009, Greenberg et al., 2009, Cohen-Cory et al., 2010). Thus, this versatile signaling protein is utilized at many stages of life to accomplish diverse tasks in the development, maintenance, and plasticity of the brain. In addition, reduced BDNF expression has been identified in many neurodegenerative diseases, and augmenting BDNF signaling has received support as a potential therapeutic strategy (see Zuccato and Cattaneo, 2009, Nagahara and Tuszynski, 2011).

BDNF exerts many of its effects by binding to the receptor tyrosine kinase TrkB and triggering an intracellular kinase cascade (see Miller and Kaplan, 2001, Chao, 2003, Reichardt, 2006). BDNF can also exert effects on cells via the pan-neurotrophin receptor p75^NTR, a member of the TNF receptor super family that binds pro-BDNF with much higher affinity than the mature form of BDNF, indicating that the processing of BDNF can be an important regulatory step (see Nykjaer et al., 2005, Underwood and Coulson, 2008, Barker, 2009, Greenberg et al., 2009, Teng et al., 2010). Both TrkB and p75 are expressed throughout the developing CNS (Buck et al., 1988, Yan and Johnson, 1988, Merlio et al., 1992). Expression of p75^NTR becomes restricted primarily to basal forebrain cholinergic and striatal neurons in the adult forebrain (see Yan and Johnson, 1988, Kiss et al., 1988, Koh et al., 1989, Sobreviela et al., 1994, Lee et al., 1998), while TrkB remains widely expressed in the adult forebrain (Merlio et al., 1992, Valenzuela et al., 1993). Forebrain-specific mutants of BDNF or TrkB share many similar phenotypes and altering BDNF or TrkB in cultured hippocampal and cortical neurons leads to similar outcomes. These data together suggest that BDNF exerts many of its cortical and hippocampal effects in the intact, healthy brain via TrkB activation (see also Conover and Yancopoulos, 1997, McAllister et al., 1999).

Both BDNF and TrkB null mutant mice lose specific populations of sensory neurons through apoptotic cell death, indicating that BDNF is necessary for the survival of a subset of sensory neurons in the peripheral nervous system (PNS) (Klein et al., 1993, Ernfors et al., 1994, Jones et al., 1994, Huang and Reichardt, 2003). In contrast, only minor cell losses have been reported in the CNS of the null mutants (see Alcantara et al., 1997, Minichiello et al., 1999). However, the essential roles of BDNF and TrkB cannot be fully evaluated in null mutant mice, because they are retarded in their postnatal growth and die within the first few weeks of life, a period during which there is extensive neuronal differentiation, cell death, and synapse formation in the CNS. To circumvent the neonatal lethality of BDNF and TrkB null mutations and allow the study of CNS requirements for BDNF and TrkB signaling, several groups have used the Cre/lox system to engineer conditional BDNF and TrkB mutants that are viable into adulthood, and these mice have been useful in defining the functions of this signaling system in the forebrain (Minichiello et al., 1999, Xu et al., 2000a, Xu et al., 2000b, Rios et al., 2001, Vyssotski et al., 2002, Gorski et al., 2003a, Gorski et al., 2003b, Zörner et al., 2003, Baquet et al., 2004, Chen et al., 2005). By selective deletion of BDNF or TrkB in different cell populations using tissue-specific promoters or viruses to direct Cre recombinase expression, it is possible to define the precise pathways in which they act (e.g. Rios et al., 2001, Eisch et al., 2003, Monteggia et al., 2004, Heldt et al., 2007, Unger et al., 2007, Li et al., 2008, Graham et al., 2009, Lobo et al., 2010). Additionally, it is possible to distinguish the developmental vs. maintenance roles of BDNF and TrkB in the CNS by using strategies including antisense oligonucleotides, early-onset vs. late-onset promoters to direct expression of Cre recombinase, or by using a mutant version of TrkB that can be inhibited with a drug, (e.g. see Ma et al., 1998, Mizuno et al., 2000, Luikart et al., 2005, Chen et al., 2005, Chan et al., 2006, Johnson et al., 2008). The combination of these types of studies continues to shed light on the precise spatial and temporal roles of BDNF–TrkB signaling.

One of the fascinating aspects of the BDNF–TrkB signaling system is its relevance to a huge variety of mammalian behaviors, including learning and memory, depression, obesity, aggression, anxiety, and development of visual acuity (see Duman and Monteggia, 2006, Chen et al., 2006, Chen et al., 2008, Lu et al., 2008, Minichiello, 2009, Heimel et al., 2010, Castren and Rantamäki, 2010, Rios, 2011, Rosas-Vargas et al., 2011). Given the widespread expression of BDNF and TrkB in the CNS, their involvement in so many aspects of mammalian life is not surprising. However, the precise neural circuitry in which BDNF and TrkB act to influence specific behaviors is largely unknown. BDNF can potentially signal in anterograde, autocrine, paracrine or the classic retrograde manner (see Altar et al., 1997, Altar and DiStefano, 1998, Nawa and Takei, 2001), thus analysis of both ligand and receptor requirements is essential for understanding signaling direction in any given behavior.

Many of the analyses studying BDNF–TrkB signaling in behavior have relied upon null mutants in which this signaling pathway is affected during development as well as in the adult. There are low levels of BDNF expression in most of the CNS at birth and levels rise during the first few weeks of life, reaching maximum levels in young adulthood (Maisonpierre et al., 1990, Schoups et al., 1995, Katoh-Semba et al., 1998). It is important to determine whether BDNF–TrkB signaling has requirements for maintenance and plasticity of mature brain circuitry that are distinct from those during development. Here, we have performed both behavioral and anatomical analyses of an early adult-onset forebrain-specific BDNF mutant mouse. We find compelling similarities with an embryonic-onset forebrain-specific BDNF mutant mouse we previously described (Gorski et al., 2003a, Gorski et al., 2003b), suggesting that sustained adult BDNF expression is essential for maintenance of some of the same brain circuitry that is sensitive to the loss of BDNF during development.