Research ReportChanges in attack behavior and activity in EphA5 knockout mice
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.
Section snippets
Increased body weight in EphA5 knockout mice
EphA5 knockout mice displayed significantly higher body weight compared to their wild-type littermates prior to any behavioral testing (F(1,94) = 53.25, p < 0.05), Fig. 1). The difference in body weight between the knockout mice and wild-type littermates was persistent throughout the entire behavioral tasks.
Altered shock-induced target-biting in EphA5 knockout mice
Under baseline conditions, mice exhibited three distinct rates of target-biting, a high post-shock rate (bin 1), an intermediate inter-shock interval rate, (bin2–7) and suppressed rate during
Discussion
Our observations demonstrate that EphA5 knockout mice have an increase in tail shock-induced target-biting in the target-biting paradigm, but a decrease in the number of bites and the latency of initiation the first attack in the resident–intruder test. EphA5 knockout mice did not show changes in their locomotor activity after encountering the intruders while their wild-type littermates exhibited significantly increased activity in their home cage after the encounter. At the neurochemical
Subjects and genotyping
EphA5 animals were obtained from Regeneron Pharmaceutical (Tarrytown, New York, USA). The generation of these knockout mice has been described previously (Feldheim et al., 2004). The line was maintained in our colony with EphA5 heterozygous knockout mice used for breeding. All mice were viable and fertile and appeared to be in good health. The genotype of mice was confirmed by polymerase chain reaction (PCR) of genomic DNA obtained from tails prior to the beginning of testing. The three primers
Acknowledgments
We would like to thank Dr. Z. Hu and Dr. B. Liou for training and assistance in genetic screening of mice and Dr. A. Halladay and L. Michna for training in behavioral paradigms. This work was supported by the RO1-DA11480, Johnson & Johnson, Busch Biomedical Research Grant, and Michael J. Fox foundation for Parkinson's Research.
References (60)
- et al.
The cytoplasmic domain of the ligand ephrinB2 is required for vascular morphogenesis but not cranial neural crest migration
Cell
(2001) - et al.
Complementary gradients in expression and binding of ELF-1 and Mek4 in development of the topographic retinotectal projection map
Cell
(1995) - et al.
Interaction of nitric oxide and serotonin in aggressive behavior
Horm. Behav.
(2003) - et al.
In vitro guidance of retinal ganglion cell axons by RAGS, a 25 kDa tectal protein related to ligands for Eph receptor tyrosine kinases
Cell
(1995) - et al.
Knockout mice reveal opposite roles for serotonin 1A and 1B receptors in prepulse inhibition
Neuropsychopharmacology
(2000) - et al.
Alterations within the endogenous opioid system in mice with targeted deletion of the neutral endopeptidase (‘enkephalinase’) gene
Regul. Pept. Lett.
(2000) - et al.
Eph receptors and ligands comprise two major specificity subclasses and are reciprocally compartmentalized during embryogenesis
Neuron
(1996) - et al.
Regulation of hippocampal synaptic plasticity by the tyrosine kinase receptor, REK7/EphA5, and its ligand, AL-1/Ephrin-A5
Mol. Cell. Neurosci.
(1998) - et al.
Brain structures and neurotransmitters regulating aggression in cats: implications for human aggression
Prog. Neuro-psychopharmacol. Biol. Psychiatry
(2001) - et al.
Neurochemical and behavioral deficits consequent to expression of a dominant negative EphA5 receptor
Brain Res. Mol. Brain Res.
(2004)
Nuk controls pathfinding of commissural axons in the mammalian central nervous system
Cell
Low cerebrospinal fluid 5-hydroxyindoleacetic acid concentration differentiates impulsive from nonimpulsive violent behavior
Life Sci.
Differential effect of Fyn tyrosine kinase deletion on offensive and defensive aggression
Behav. Brain Res.
Pleiotropic contributions of nitric oxide to aggressive behavior
Neurosci. Biobehav. Rev.
The influence of sex and social isolation housing on pre-and postsynaptic 5-HT1A receptors
Brain Res.
Neuropharmacology of brain-stimulation-evoked aggression
Neurosci. Biobehav. Rev.
Behavioral stress response of genetically selected aggressive and nonaggressive wild house mice in the shock-probe/defensive burying test
Pharmacol. Biochem. Behav.
Postnatal development of a GABA deficit and disturbance of neural functions in mice lacking GAD65
Brain Res.
Androgen-dependency of aggressive target-biting and paired fighting in male mice
Physiol. Behav.
The Eph family receptors and ligands
Pharmacol. Ther.
Serotoninergic dysfunction in the 47, XYY syndrome
Biol. Psychiatry
Multiple ephrins regulate hippocampal neurite outgrowth
J. Comp. Neurol.
The effects of repeated administration of fluprazine on target biting and intruder-evoked attacks
Psychopharmacology (Berl.)
Dual action of a ligand for Eph receptor tyrosine kinases on specific populations of axons during the development of cortical circuits
J. Neurosci.
Brain serotonin dysfunction accounts for aggression in male mice lacking neuronal nitric oxide synthase
Proc. Natl. Acad. Sci. U. S. A.
Neural connections of the anterior hypothalamus and agonistic behavior in golden hamsters
Brain Behav. Evol.
Loss-of-function analysis of EphA receptors in retinotectal mapping
J. Neurosci.
Serotonin and aggressive behavior in rodents and nonhuman primates: predispositions and plasticity
Eur. J. Pharmacol.
Accumbal dopamine and serotonin in anticipation of the next aggressive episode in rats
Eur. J. Neurosci.
Aggression heightened by alcohol or social instigation in mice: reduction by the 5-HT(1B) receptor agonist CP-94,253
Psychopharmacology (Berl.)
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