TNF-α and its receptors modulate complex behaviours and neurotrophins in transgenic mice
Introduction
Tumour necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine showing biological effects in the central nervous system (CNS) (Stellwagen et al., 2005, McAfoose and Baune, 2009, Clark et al., 2010, Baune et al., 2012) In the CNS, TNF-α is produced by the resident immune cells of the CNS, the microglia and astrocytes, as well as by certain neuronal populations (Li et al., 2008, McCoy and Tansey, 2008). This cytokine is shown to cross the blood–brain barrier (BBB) in conditions of systemic inflammation (Pan and Kastin, 2002) and is known to play a role in activating the hypothalamic pituitary adrenal (HPA) axis by stimulating the corticotrophin-releasing hormone and increasing levels of corticosterone (Bernardini et al., 1990). High levels of TNF-α in the dorsolateral prefrontal cortex have also been linked to major depressive disorder (Dean et al., 2010) and increased expression of TNF-α were also observed in murine models of depressive like behaviour and chronic mild stress (You et al., 2011, Kaster et al., 2012).
Beyond its involvement in various CNS pathologies, TNF-α may be is required for maintaining certain physiological processes within the CNS (Stellwagen et al., 2005). TNF-α has been shown to play a role in mediating synaptic scaling (Stellwagen et al., 2005), and was found to be essential to early cognitive development (McAfoose et al., 2009). Constitutive levels of TNF-α are linked to increased neurogenesis (Iosif et al., 2006) and TNF-α is also shown to influence the expression of neurotrophins, such as nerve growth factor (NGF) (Takei and Laskey, 2008) and brain derived neurotrophic factor (BDNF) (Golan et al., 2004). Indeed, modulation of neurotrophins may explain the finding that young adult mice that lack TNF-α (TNF−/− mice) exhibit cognitive impairment (Baune et al., 2008). Furthermore, TNF-α can play an integral part in modulating anxiety like behaviour as well as in fear conditioning (Yamada et al., 2000, Simen et al., 2006).
TNF-α signals through two main receptors, TNF-R1 and TNF-R2, which are thought to have disparate functions, with suggestions that activation of TNF-R1 is neurodegenerative, whilst activation of TNF-R2 is neuroprotective (Shu et al., 1996, Kassiotis and Kollias, 2001, Fontaine et al., 2002). This is because signalling through the TNF-R1 pathway, which primarily occurs via soluble TNF-α, initiates the activation of caspases leading to apoptosis, due to the presence of a cytoplasmic ‘death domain’. In the brain, TNF-R1 is widely expressed by astrocytes, oligodendrocytes and microglia, whereas only microglial cells express TNF-R2 (Dopp et al., 1997). In immunologically unchallenged conditions TNF-R1 appears to be more abundantly expressed by neurons in the brain while basal levels of TNF-R2 are expressed only in the cortex, cerebellum and tegmentum (Bette et al., 2003). In contrast, activation of the TNF-R2 pathway, which primarily occurs via transmembrane TNF-α, promotes cell survival, through inhibition of caspase activation (Horiuchi et al., 2010). Intriguingly, the distinction between these two signalling pathways does not appear to be as clear cut as previously thought, with an earlier study demonstrating that TNF−/−, TNF-R1−/− and TNF-R2−/− mice all exhibited cognitive deficits (Baune et al., 2008), suggesting that signalling through the receptors is not exclusively neurodegenerative or neuroprotective.
Therefore, to further explore the role of TNF-R1 and TNF-R2 signalling in behavioural responses, we have performed an extensive behavioural battery assessing cognition-like behaviour (learning and memory), social interactions, exploratory activity, anxiety-like behaviour and depression-like behaviour in young adult (12-week old) TNF−/−, TNF-R1−/− and TNF-R2−/− transgenic mice. Furthermore, to determine whether behavioural changes in these transgenic mice relate to underlying changes in neurotrophins, these parameters were also investigated. While previous studies have investigated the effects of genetic deletion of TNF-α and its receptors on cognition-like (Baune et al., 2008) and anxiety-like behaviours (Yamada et al., 2000, Simen et al., 2006), this is the first study to provide a comprehensive description of a wider range of behavioural phenotypes, including cognition-, anxiety- and depression-like behaviours extending into sociability and exploratory behaviour, governed by TNF-α and its receptors, while considering molecular mechanisms of neurotrophins and neurogenesis expression to influence such behaviour.
Section snippets
Mouse strains
The gene-targeted C57BL/6 mouse strain deficient for TNF-α (TNF−/−) (n = 14, seven male, seven female mice) was generated on a genetically pure C57BL/6 background as described previously (Korner et al., 1997) and has been bred in-house for over five generations. The C57BL/6 (WT) mice (Jackson stock number: 000664) were purchased from the University of Adelaide breeding facility (n = 21, 10 male, 11 female). TNF-R1−/− (Jackson stock number: 003242) (Peschon et al., 1998) and TNF-R2−/− mice (Jackson
Mean weight, age and sex
Mean age of all animals used was 12 weeks at the start of behavioural testing. The average weight of mice was 24.0 ± 1.4 g for male mice and 18.0 ± 1.5 g for female mice of all strains and no significant differences were observed between strains as analysed by Kruskal–Wallis test.
To minimise potential gender-specific effects in behaviour that could arise from genetic deletion of TNF-α and TNF-α receptor related genes, roughly equal numbers of male and female mice were used in the present study. No
Discussion
In this study we have shown that TNF−/− mice displayed impairments in memory in the Barnes maze and Y-maze, while TNF-R2−/− mice showed good memory but slow learning in these tests. TNF-R2−/− and TNF−/− mice also showed low levels of anxiety like behaviour. All strains of mice demonstrated a significant preference for social situations over non-social ones, however only WT and TNF−/− mice showed a significant preference for social novelty. Analysis of neurotrophin levels in the prefrontal
Role of funding source
This study is supported by the National Health and Medical Research Council Australia (APP1003788 to BTB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Conflict of interest
None declared.
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