Elsevier

Psychoneuroendocrinology

Volume 85, November 2017, Pages 134-141
Psychoneuroendocrinology

Deletion of Dlk2 increases the vulnerability to anxiety-like behaviors and impairs the anxiolytic action of alprazolam

https://doi.org/10.1016/j.psyneuen.2017.08.015Get rights and content

Highlights

  • Dlk2-knockout mice were generated and their emotional behavior was evaluated.

  • Deletion of Dlk2 increased anxiety-like, depressive-like behaviors, and vulnerability to restraint stress.

  • Crh gene expression in the PVN, and Nr3c1 and Fkbp5 gene expression in the HIPP were affected.

  • Gabra2 gene expression was significantly lower in Dlk2–/–mice.

  • Alprazolam failed to produce an anxiolytic-like action in Dlk2–/– mice.

Abstract

The purpose of this study was to evaluate the role of the non-canonical DLK2 NOTCH ligand in the regulation of emotional behavior. To this aim, anxiety and depressive-like behaviors were examined in Dlk2 knock-out (Dlk2–/–) and its corresponding wild-type (WT) mice. Furthermore, relative gene expression analyses of corticotropin releasing hormone (Crh) in the paraventricular nucleus (PVN), glucocorticoid receptor (NR3C1) and FK506 binding protein 5 (FKBP5) in the hippocampus (HIPP), and the transcription factors Hes1, Hes5 and Hey1 in the PVN, HIPP and amygdala (AMY) were carried out in Dlk2–/– and WT mice under basal conditions and after exposure to restraint stress. The anxiolytic action of alprazolam and the relative gene expression levels of the GABA-A alpha 2 and gamma 2 receptor subunits (Gabra2 and Gabrg2) were also evaluated in the HIPP and AMY of WT and Dlk2–/– mice.

The results reveal that deletion of Dlk2 increased anxiety and depressive-like behaviors and altered the vulnerability to restraint stress on Crh gene expression in the PVN, Nr3c1 and Fkbp5 gene expression in the HIPP, and Hes1, Hes5 and Hey1 gene expression in the PVN, HIPP and AMY. Interestingly, the administration of alprazolam failed to produce an anxiolytic action in Dlk2–/– mice. Indeed, Gabra2 and Gabrg2 gene expression levels were significantly affected under basal conditions and after stress exposure in Dlk2–/– mice compared with WT mice.

In conclusion, the results suggest that DLK2 plays an important role in the regulation of emotional behaviors and relevant targets of the stress axis, NOTCH pathway and GABAergic neurotransmission. In addition, the deletion of Dlk2 blocked the anxiolytic response to alprazolam. Future studies are needed to determine the relevance of DLK2 as a potential therapeutic target for the treatment of neuropsychiatric disorders with anxiety or depressive-like behaviors.

Introduction

The NOTCH system is an evolutionary conserved signaling pathway identified as a critical regulator of neurogenesis in the adult brain (Imayoshi and Kageyama, 2011). In mammals, NOTCH signaling is triggered by the interaction of at least one of the four membrane receptors (NOTCH1–4) with one or more of the five JAGGED of DELTA canonical ligands (JAGGED1, JAGGED2, DLL1, DLL3, and DLL4) (Kageyama and Ohtsuka, 1999, D'Souza et al., 2010). NOTCH receptors and their ligands are transmembrane proteins belonging to the epidermal growth factor (EGF)-like family. The interaction induces metalloproteinase-mediated and γ-secretase-mediated cleavage of the NOTCH receptor releasing an active NOTCH intracellular domain (NICD) that translocates to the nucleus and forms a complex with CBF-1/RBP-J, mediating the transcription of target genes, for instance, the expression of the basic loop helix transcription factors Hes1, Hes5 and Hey1 (D'Souza et al., 2008). Recently, two EGF-like proteins highly related to the NOTCH ligand family, named Delta-like 1 (DLK1) and Delta-like 2 (DLK2), were identified as non-canonical NOTCH ligands, based on the fact that they interact with NOTCH receptors, despite lacking the DSL domain characteristic of the canonical ligands. Although the precise function of these non-canonical ligands still remains unclear, DLK1 and DLK2 act as NOTCH receptor antagonists (Baladron et al., 2005, Sanchez-Solana et al., 2011, Traustadottir et al., 2016).

Among the different functional implications of the NOTCH signaling pathway, numerous evidences emphasize its crucial involvement in cell differentiation and proliferation processes (Falix et al., 2012), playing a key role in tumorigenesis, carrying important therapeutic implications (Shao et al., 2012, Espinoza and Miele, 2013, Gu et al., 2016, Liu et al., 2016). In addition, in the central nervous system (CNS), the NOTCH pathway contributes to the maintenance of an adequate balance between neurogenesis and gliogenesis (Snyder et al., 2012), to the regulation of synaptic plasticity (Alberi et al., 2011), and to neuron survival (Hitoshi et al., 2004, Mizutani et al., 2007). Indeed, recent studies described how the NOTCH pathway is involved in the regulation of different brain functions, such as learning, memory (Costa et al., 2003, Conboy et al., 2007, Yoon et al., 2012, Sargin et al., 2013, Ding et al., 2016) or emotional behavior (Guo et al., 2009, Wang et al., 2012, Dias et al., 2014, Monsalve et al., 2014, Sun et al., 2016). However, additional studies are still needed to elucidate the precise neurobiological mechanisms underlying NOTCH-mediated actions.

DLK2 is a transmembrane protein highly homologous to the previously known DLK1, characterized by a widespread expression in different mouse tissues (Nueda et al., 2007). Besides its role as a non-canonical NOTCH ligand inhibiting NOTCH signaling, there is very scarce information about the functional role of DLK2. Interestingly, DLK2 is widely distributed in different brain areas such as the arcuate nucleus, the hypophysis, the dorsal raphe, the amygdala, the hippocampus and the prefrontal cortex (Gregg et al., 2010, Monsalve et al., 2014, Bonthuis et al., 2015, Carithers and Moore, 2015, Lizio et al., 2015). Since all these regions are involved in the regulation of stress, emotional response, learning, or memory, it is tempting to suggest that DLK2 may play a relevant role in the regulation of these behaviors.

Mice deficient for the Dlk2 gene (Dlk2−/− mice) were generated and used to evaluate the relevance of this gene in emotional behavior. Basic behavioral aspects related with motor activity (open field test), anxiety-like (light-dark box test, elevated plus maze test) and depressive-like behavior (novelty suppressed feeding test) were performed in WT and Dlk2–/– mice. The neurobiological changes induced by challenging animals to an acute stressful stimulus (restraint stress) and the pharmacological response to the anxiolytic benzodiazepine (alprazolam) were evaluated. Analyses of the relative gene expression levels of corticotropin releasing hormone (Crh) in the paraventricular nucleus (PVN), glucocorticoid receptor (Nr3c1), FK506 binding protein 5 (Fkbp5) in the hippocampus (HIPP), and the transcription factors Hes1, Hes5 and Hey1 in PVN, HIPP and amygdala (AMY) were carried out in Dlk2–/– and WT mice under basal conditions and after exposure to restraint stress. In addition, gamma-aminobutyric acid (GABA) A receptor, subunit alpha-2 (Gabra2) and subunit gamma-2 (Gabrg2), gene expression levels were also evaluated in the amygdala (AMY) of Dlk2−/− and WT mice under basal conditions and after exposure to restraint stress.

Section snippets

Generation of Dlk2 knockout mice

To generate Dlk2 knockout mice we designed a targeting strategy consisting in the insertion in this locus of a single distal loxP site within the intron 1 and a loxP site together with a flippase recognition target (FRT) flanked neomycin selection cassette within the intron 4 (Fig. S1). Three of the confirmed properly recombined embryonic stem (ES) cell clones (from S129P2/Ola background) were then injected into blastocysts (C57BL6/J background), aiming to generate Dlk2 constitutive knockout

Motor activity-open field

No significant differences between Dlk2−/− mice and their corresponding control group were observed in any of the parameters analyzed (Fig. 1) (periphery distance travelled Student’s t-test, t = 0.570, 28df, p = 0.573; center distance travelled Student’s t-test, t = −1.795, 28df, p = 0.083; total distance travelled Student’s t-test, t = −0.782, 28df, p = 0.441; periphery speed Student’s t-test, t = −1.452, 28df, p = 0.158; center speed Student’s t-test, t = −1.452, 28df, p = 0.158; total speed Student’s t-test, t = 

Discussion

The results of the present study point to DLK2 as playing a major role in the regulation of emotional behaviors. This assumption is based on the following facts: 1) Deletion of Dlk2 increased vulnerability to anxiety-like (light-dark box and elevated plus maze paradigms) and depressive-like behaviors (novelty suppressed feeding test); 2) Crh, Nr3c1 and Fkbp5 gene expression levels were altered in Dlk2–/– mice under both basal and restraint stress conditions compared with WT mice; 3) Hes1, Hes5

Author contribution

JL and JM were responsible for the study concept and design. FN and MSGG contributed to the acquisition of animal data. FN, MSGG, JL and JM assisted with data analysis and interpretation of findings. JL designed and directed the generation of the animals. FN drafted the manuscript. JL and JM provided critical revision of the manuscript for important intellectual content. All authors critically reviewed content and approved the final version for publication.

Conflict of interest

All authors state that they have no biomedical financial interest or potential conflicts of interest.

Acknowledgments

This research was supported by “Instituto de Salud Carlos III” (RTA, RD16/0017/0014, Fondos FEDER), “Plan Nacional sobre Drogas” (PNSD, 2016/016), ‘Ministerio de Economía y Competitividad’ (FIS, PI14/00438), and ‘Junta de Comunidades de Castilla-La Mancha’ (PCI08-0134-6712, PEI-1-2014-033, with FEDER funds).

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      Although unlikely, we cannot rule out the possibility that one of those alleles accounts for the behavioral differences noted, rather than Dlk1. This alternative hypothesis is weakened, however, by the fact that Dlk2 knockout mice also show behavioral abnormalities related to anxiety disorders [73]. Dlk2 is located at mouse chromosome 17, so it does not share any nearby alleles with Dlk1.

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    Both authors share equal credit for the direction of this work.

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