Changes in attack behavior and activity in EphA5 knockout mice

Abstract

During development, Eph tyrosine kinase receptors and their ephrin ligands function as axon guidance molecules while, in adults, these molecules appear to be involved in the regulation of neural plasticity and emotion. The absence of EphA5 receptor mediated forward signaling may cause alterations in connectivity of neural networks and boundary formation during development, including central monoaminergic systems. In the present studies, we demonstrated altered aggressive responses by animals lacking functional EphA5 receptors. These behavioral changes were accompanied by altered concentrations of serotonin (5-HT) and the metabolite, 5-HIAA, in the hypothalamus. The changes of serotonin activity in hypothalamus also result in increase of body weight in EphA5 knockout mice. Furthermore, EphA5 knockout mice exhibited a significant decrease in activity levels following exposure to naïve intruders in their home cages. We conclude that the EphA5 receptor may be involved in mediation of aggressive behavior regulated, in part, by hypothalamic serotonin.

Introduction

The EphA5 receptor is a member of the Eph receptor tyrosine kinase family. Eph receptor tyrosine kinases and their corresponding ligands, ephrins, comprise the largest group of receptor tyrosine kinases with at least eight ligands in vertebrates (Wilkinson, 2001, Zhou, 1998). In the human genome, 13 Eph receptors have been identified and are found to be distributed in three separate chromosomes (Kullander et al., 2001). Based on the structural homology and the binding preference, ephrins are classified into two groups, ephrin-A and ephrin-B. Ephrin-A ligands generally bind to EphA receptors, whereas ephrin-B ligands bind to EphB receptors (Himanen et al., 2004). There are some exceptions to this general rule; e.g., the EphA4 receptor which can bind to both ephrin-A and ephrin-B ligands. A-type ephrins are anchored to the membrane through a glycosylphosphatidylinositol (GPI) linkage and B-type ephrins have both transmembrane and cytoplasmic regions (Flanagan and Vanderhaeghen, 1998, Gale et al., 1996).

The function of the EphA5 receptor is best characterized as an axon guidance molecule during neural development (Cheng et al., 1995, Yue et al., 2002). The EphA5 receptor and its ligand act as a repellant cue that prevents axons from entering inappropriate territories, thus restricting the cells to specific pathways during the migratory process (Wilkinson, 2001). During neural development, Eph receptors and their ligands are expressed in the projecting and target sites, respectively (Castellani et al., 1998, Gale et al., 1996, Gao et al., 1998a, Stein et al., 1999, Zhang et al., 1996). For example, in the case of hippocamposeptal projections, EphA5 receptors are expressed in a gradient with the lateral hippocampus expressing low levels and the medial hippocampus expressing high levels of the receptor. At the target site, the lateral septum, ephrin-A5 is expressed with a complementary gradient such that the dorsomedial septum expresses low levels and the ventrolateral septum expresses high levels of this ligand (Zhang et al., 1996). During embryogenesis, the EphA5 transcript is highly expressed in the cortical plate (Castellani et al., 1998). It is also expressed in cortex, hippocampus, medial thalamus and the septum of the developing brain. This receptor is moderately expressed in other brain regions, including hypothalamus and amygdala (Gao et al., 1998a).

At the cellular level, the binding of ephrin-A5 with receptor-expressing neurons results in different consequences depending on the cell type. It has been demonstrated that this interaction causes inhibition of the neurite outgrowth of hippocampal, striatal, retinal, and cortical neurons, while it enhances the neurite outgrowth of sympathetic neurons and stimulates neurite sprouting of cortical neurons in vitro (Brownlee et al., 2000, Gao et al., 2000, Gao et al., 1998a, Gao et al., 1996). At the circuit level, overexpression of a truncated form of EphA5 receptor resulted in a miswiring of the hippocamposeptal pathway and corpus callosum connections in vivo (Yue et al., 2002). In particular, medial hippocampal neurons with high expression level of the EphA5 receptor projected to both the ventral and lateral part of the target site while lateral hippocampal neurons with relatively low EphA5 receptor expression did not exhibit any obvious alteration in their projection pattern. Taken together, the EphA5 receptor and its ligands serve as repulsive axon guidance cues in the developing brain. Their interaction triggers growth cone collapse and inhibits the neurite outgrowth in vitro. Furthermore, abnormal expression of these molecules results in the disruption of axonal pathfinding and mid-line crossing in vivo (Henkemeyer et al., 1996, Hu et al., 2003, Yue et al., 2002).

Details of how the binding of the Eph receptors and their ligands inhibits neurite outgrowth are yet to be determined. Gale and Yancopoulos (1997) provided evidence for the collapse of actin cytoskeletal structure within the growth cone following the activation of ephrin-induced signaling. Signal transduction induced by Eph–ephrin binding requires the autophosphorylation of the Eph receptor (Drescher et al., 1995, Meima et al., 1997). This event occurs predominately based on the cell−cell contact. Soluble forms of ephrins can bind to Eph receptors, but do not trigger autophosphorylation unless the receptors are artificially assembled. Furthermore, this receptor−ligand system can activate the intracellular signaling pathways not only via the activation of Eph receptor, but also by clustering of ephrins. Recent studies have shown that reverse signaling induced by ephrin clustering affects commissural formation in the forebrain as well as angiogenic remodeling (Adams et al., 2001, Henkemeyer et al., 1996, Kullander et al., 2001).

The presence of a phosphorylated form of EphA5 receptor in the adult brain leads to the speculation about possible roles in synaptic plasticity (Gerlai et al., 1999). By infusing EphA5 receptor agonist/antagonist proteins into the hippocampus, Gerlai et al. (1999) showed that activation of the EphA5 receptor enhances hippocampal-dependent behavioral tasks whereas the inactivation of the EphA5 receptor impairs these functions. Specifically, animals exhibited elevated fear responses consequent to shock exposure following EphA5 receptor agonist infusion. These behavioral changes were also accompanied by alterations in long-term potentiation suggesting the role of EphA5 receptor in synaptic plasticity (Gao et al., 1998b). In addition, Halladay et al. (2004) showed that animals expressing a truncated EphA5 receptor exhibited learning deficits in striatal-dependent tasks. These behavioral changes were associated with changes in monoaminergic activities in striatum suggesting a possible role of EphA5 receptor in striatal functions. Taken together, activation of EphA5 receptor and its ligand may be involved in synaptic plasticity in the adult nervous system.

The expression of EphA5 receptor is elevated in hippocampus, striatum, hypothalamus, and amygdale in the adult brain (Gerlai et al., 1999). In this study, we asked whether the absence of EphA5 receptor mediated forward signaling can affect brain neurochemistry and how the altered neurochemistry might affect the aggressive behaviors mediated by hypothalamus in adult animals. Offensive aggression was assessed using the resident–intruder paradigm. Offensive aggression is predominantly a testosterone-dependent behavior and is manifest as the attack behavior of a resident subject against an intruder (Wagner et al., 1979). This type of offensive aggression can also be modulated by serotonin activity and drugs (Chiavegatto et al., 2001, Fish et al., 1999, Lyons et al., 1999, Miczek et al., 1998). Genetic manipulations that target serotonin-related genes, and on genes that affect serotonin receptor numbers also can change this form of aggression in rodents (Chiavegatto et al., 2001, Chiavegatto and Nelson, 2003, Dulawa et al., 2000, Fischer et al., 2000, Liu et al., 2007, Nelson et al., 2006, Saudou et al., 1994, Schiller et al., 2006, Stork et al., 2000, Wersinger et al., 2007). We also assessed defensive aggression by using the target-biting paradigm. It has been shown that high serotonergic activity dampens both defensive aggression in animals and violent crime in humans and, conversely, reduced serotonergic activity is associated with high levels of aggression (Bioulac et al., 1980, Golden et al., 1991, Lidberg et al., 1985, Linnoila et al., 1983, Virkkunen et al., 1987).

Our results showed that EphA5 knockout mice exhibit an increase in shock-induced target-biting but a decrease in offensive aggression in the resident–intruder paradigm. The escalated levels of 5-HT and 5-HIAA found in hypothalamus may have contributed the decrease in offensive aggression in knockout mice. Interestingly, EphA5 knockout mice showed significantly higher body weight than the controls. This increase in body weight is likely attribute to the change in serotonin metabolism in hypothalamus. Moreover, EphA5 knockout mice exhibited decreased motor activity immediately following the resident–intruder test in the same context. We concluded that the absence of EphA5 receptor-induced signaling results in alterations of aggressive behaviors and these behavioral changes are accompanied by changes in serotonergic activity in the hypothalamus.