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- Materials and methods
EphA4 receptor (EphA4) tyrosine kinase is an important regulator of central nervous system development and synaptic plasticity in the mature brain, but its relevance to the control of normal behavior remains largely unexplored. This study is the first attempt to obtain a behavioral profile of constitutive homozygous and heterozygous EphA4 knockout mice. A deficit in locomotor habituation in the open field, impairment in spatial recognition in the Y-maze and reduced probability of spatial spontaneous alternation in the T-maze were identified in homozygous EphA4−/− mice, while heterozygo us EphA4+/− mice appeared normal on these tests in comparison with wild-type (WT) controls. The multiple phenotypes observed in EphA4−/− mice might stem from an underlying deficit in habituation learning, reflecting an elementary form of nonassociative learning that is in contrast to Pavlovian associative learning, which appeared unaffected by EphA4 disruption. A deficit in motor coordination on the accelerating rotarod was also demonstrated only in EphA4−/− mice – a finding in keeping with the presence of abnormal gait in EphA4−/− mice – although they were able to improve performance over training. There was no evidence for substantial changes in major neurochemical markers in various brain regions rich in EphA4 as shown by post-mortem analysis. This excludes the possibility of major neurochemical compensation in the brain of EphA4−/− mice. In summary, we have demonstrated for the first time the behavioral significance of EphA4 disruption, supporting further investigation of EphA4 as a possible target for behavioral interventions where habituation deficits are prominent.
The Eph receptor A4 (EphA4) is a member of the Eph family of receptor tyrosine kinases (Ephs). Ephs and their cell surface-associated ephrin ligands are important regulators of central nervous system (CNS) development; they are involved in the guidance of axonal growth and establishment of neural connectivity (Egea & Klein 2007; Pasquale 2008). In the mature brain, Ephs regulate neuron–glia communication and play a role in synaptic plasticity (Klein 2009; Murai & Pasquale 2011). Among the subtypes, EphA4 shows widespread expression throughout the CNS starting from early development and persisting in the mature brain (Greferath et al. 2002). In the adult hippocampus, EphA4 is expressed on dendritic spines of pyramidal neurons and axon terminals (Bouvier et al. 2008; Carmona et al. 2009; Filosa et al. 2009; Murai et al. 2003; Tremblay et al. 2007), where it interacts with ephrin-A3 to shape dendritic spine morphology and influences synaptic plasticity (Carmona et al. 2009; Filosa et al. 2009; Murai et al. 2003). Eph dysfunction might be relevant to neuropsychiatric and neurodegenerative diseases (Inoue et al. 2009; Yamaguchi & Pasquale 2004) characterized by abnormal dendritic spines in the hippocampus and cortex (Glantz & Lewis 2000; Kaufmann & Moser 2000). Specifically, a genetic mouse model of Alzheimer's disease overexpressing human amyloid-β protein precursor was accompanied by reduced hippocampal EphA4 expression (Simón et al. 2009), suggesting that aberrant EphA4-mediated signaling might contribute in part to the memory deficits in this model.
However, the functional significance of EphA4 in normal behavior and cognition remains largely unexplored. This study is the first attempt to characterize the impact of constitutive genetic deletion of EphA4 on behavior and cognition in adulthood. Five behavioral tests were performed, including a test of motor coordination using the accelerating rotarod, open field locomotor activity, anxiety-related behavior in the elevated plus maze, spontaneous alternation in the T-maze, spatial recognition memory in the Y-maze and conditioned context freezing. Performance in the last four tests is sensitive, albeit not exclusively so, to hippocampal damage. They were selected because EphA4 is not only highly expressed in the hippocampus but also involved in hippocampal synaptic plasticity, such as long-term potentiation (Grunwald et al. 2004); and therefore EphA4 may assume a role in hippocampus-dependent behavior. Both homozygous (EphA4−/−) and heterozygous (EphA4+/−) EphA4 knockout (KO) mice were tested and compared with WT littermate controls (EphA4+/+).
We demonstrate here for the first time that EphA4−/− mice are associated with reduced locomotor habituation and impaired spatial novelty detection. In addition, we report a deficit in motor coordination and balance on the accelerating rotarod, which is most likely related to altered neuronal connections in the spinal cord and impaired hind limb gaits previously demonstrated in these animals (Akay et al. 2006; Dottori et al. 1998; Kullander et al. 2001). We also found no indication of potential developmental alterations in major neurochemical markers in the brain, although the contribution of other developmental factors cannot be excluded. This study highlights a critical role of EphA4 in short-term habituation processes and novelty assessment.
- Top of page
- Materials and methods
This study showed that constitutive homozygous deletion of EphA4 yielded multiple phenotypes on (1) the open field test of spontaneous motor activity and motor habituation, (2) the Y-maze test of short-term familiarity judgment underlying spatial recognition and (3) spontaneous alternation on the T-maze , without affecting Pavlovian contextual fear conditioning. Furthermore, a deficit in motor function was detected in EphA4−/− mice, which is most likely related to the hind limb coordination problems previously identified in these animals (Akay et al. 2006; Dottori et al. 1998; Kullander et al. 2001).
Anatomical studies of EphA4−/− mice have shown that genetic deletion of EphA4 led to dysfunctional central pattern generators in the spinal cord responsible for the coordination of limb alternation underlying normal walking (Kiehn & Butt 2003; Kullander et al. 2001, 2003), as indicated by the ‘hopping gait’ in EphA4−/− mice (Dottori et al. 1998; Kullander et al. 2001) possibly attributable to an excitation/inhibition imbalance in the spinal cord (Restrepo et al. 2011). The severe deficit in rotarod performance in the EphA4−/− mice reported here is also novel, but not surprising. Despite this deficit, EphA4−/− mice nonetheless were able to demonstrate an improvement over training, suggesting that EphA4 deletion did not completely disrupt motor skill learning, which apparently requires the cerebellum (Lalonde & Strazielle 2007; Llinas & Welsh 1993). Hence, even though EphA4 is heavily expressed in the cerebellum from neonatal to adult life (Greferath et al. 2002; Karam et al. 2000; Liebl et al. 2003; Martone et al. 1997; Xiao et al. 2006) and is implicated in cerebellar wiring (Cesa et al. 2011; Karam et al. 2000), its deletion did not completely prevent cerebellum-dependent motor skill learning.
A previous study has reported that EphA4−/− mice were less active in the open field than EphA4+/− mice (Dottori et al. 1998), but examination of locomotor activity in that study was limited to 5 min, and the authors did not provide any data on WT controls for comparison. This reported phenotype is seemingly opposite to our current finding of hyperlocomotor activity in EphA4−/− mice. However, the hyperlocomotor activity phenotype did not emerge until habituation became evident in the EphA4+/− and EphA4+/+ mice, which emerged after the first 5 min and persisted until the end of the 1-h test. This observation was replicated on the second test session 24 h later. In contrast to this deficit in within-session motor habituation, habituation across test sessions as evidenced by the overall reduction of activity levels from day 1 to day 2 was not significantly altered in EphA4−/− mice. Thus, the open field locomotor activity phenotype suggests an underlying deficit in short-term, but not in long-term, locomotor habituation.
At the same time, we also observed that the EphA4−/− mice spent significantly more time in the central zone of the open field, which might be interpreted as anxiolysis (Prut & Belzung 2003); but this interpretation should be cautioned because the behavior of EphA4−/− mice in the elevated plus maze test of anxiety was not significantly altered. Additional tests of anxiety would be warranted to ascertain whether the null results in the elevated plus maze might represent a type-II error attributable to the higher level of individual variability amongst the EphA4−/− mice.
Short-term habituation is a critical factor that influences spontaneous alternation behavior in the T-maze and spatial recognition memory in the Y-maze (Honey & Good 2000; Sanderson et al. 2010). Both tests are based on the innate motivation of mice to explore novel environments, whereby the preferential exploration of a novel over a familiar environment is commonly taken as an index of spatial novelty detection (Dellu et al. 1992; Dember & Fowler 1958). Wagner's sometimes opponent process (SOP) model (Wagner 1981) describes short-term habituation as a shift from the center to the periphery of attention, such that stimuli in the periphery of attention are less able to elicit a response. It follows that the novel arm in the Y-maze and the T-maze commands more attention and therefore elicits more intense exploration than the familiarized, visited arm, which would be in the periphery of attention according to Wagner's SOP model. Short-term habituation is defined as decrement in behavioral response when a stimulus is presented repeatedly and is considered an elementary form of nonassociative learning (Thompson 2009; Thompson & Spencer 1966). In this sense, locomotor hyperactivity, disruption of alternation behavior in the T-maze and impaired spatial novelty detection observed in the Y-maze in the EphA4−/− mice may reflect a form of cognitive deficit specific to nonassociative learning. By contrast, associative learning appears to be unaffected in these animals as evidenced by intact Pavlovian fear conditioning. There is indeed evidence that performance in the T-maze and Y-maze tests primarily relies on short-term habituation but is largely independent of associative learning (Sanderson et al. 2010). Hence, a specific deficit in short-term habituation may provide a parsimonious account of the three phenotypes identified in the EphA4−/− mice in the open field as well as the Y- and T-mazes.
Although the use of a constitutive KO model does not allow one to dissect region-specific mechanisms, speculations on the involvement of relevant brain regions can guide further investigation. One plausible candidate region is the hippocampus where EphA4 is critical for the regulation of synaptic plasticity including long-term potentiation (Grunwald et al. 2004). Hippocampal lesions not only induce hyperactivity and retard habituation (Poucet 1989) but also robustly impair spontaneous alternation in the T-maze and spatial recognition memory in the Y-maze (Bannerman et al. 2004; O'Keefe & Nadel 1978). Studies of neuronal activity also suggest a specific role of the hippocampus in the detection of spatial novelty (Rinaldi et al. 2010; Zhu et al. 1997). Furthermore, several theories have linked the hippocampus to the ability to compare the present state of the world with what is expected based on retrieved memories to guide appropriate behavioral response (Gray 1982; McNaughton 2006; Vinogradova 2001). According to this concept, Gray (1982) suggested that an important function of the hippocampus is to increase arousal and attentional processing following detection of a novel stimulus and thus to enhance exploration (McNaughton 2006; Vinogradova 2001). However, the involvement of hippocampal EphA4 and in particular the contribution of hippocampal dysfunction to the behavioral phenotypes identified in EphA4−/− mice should not be overestimated, because hippocampus-dependent contextual fear conditioning remained intact in EphA4−/− mice. Instead, contextual fear conditioning seems to be regulated by another member of the EphA receptor family, namely the EphA5 subtype (Gerlai et al. 1999). Nevertheless, the role of EphA4 as a potential regulator of hippocampal functions certainly deserves further investigation by using region-specific KO models in order to examine whether disruption of EphA4 in the hippocampus alone would be sufficient to reproduce the behavioral phenotypes seen in constitutive KOs here. Another candidate brain region that warrants consideration is the striatum, which is rich in EphA4, and where EphA4 regulates the development of the spatial patterning of the striatum during maturation (Passante et al. 2008). The striatum is implicated in the regulation of locomotion and novelty-evoked behavioral activation (Berns et al. 1997; Hooks and Kalivas 1995; Rinaldi et al. 2010). Although there is evidence for a participation of the striatum in spatial novelty detection and spontaneous alternation behavior, its precise role is in need of further clarification (Cigrang et al. 1986; Hagan et al. 1983; Lalonde 2002; Roullet et al. 2001; Taghzouti et al. 1985; Thullier et al. 1996; Usiello et al. 1998). The striatum is also known for its key role in procedural and habit learning (Knowlton et al. 1996; Mishkin & Petri 1984; Yin et al. 2004), which has not been explicitly tested in this study. Nonetheless, a contribution of altered striatal function to the behavioral phenotypes of the EphA4−/− mice should be considered, although not necessarily in exclusion of a parallel hippocampal contribution in our constitutive KO model.
Here, none of the behavioral phenotypes identified in the homozygous EphA4 KOs was evident in the heterozygous KOs, suggesting that a single copy of the EphA4 gene with about 50% of normal protein expression was insufficient to significantly alter behavior in the present experiments. Furthermore, no changes in various neurochemical markers were detected across diverse brain regions rich in EphA4 protein expression. These negative findings support the suggestion that the observed behavioral effects resulted indeed from the genetic disruption of EphA4 rather than potential developmental compensation in other neurotransmitters – although the contribution of additional developmental factors cannot be excluded, which should be best addressed by temporally controlled gene deletion or local pharmacological blockade in adult mice.
In conclusion, our study shows that constitutive homozygous deletion of EphA4 leads to a distinct set of behavioral phenotypes including impaired short-term habituation and novelty detection. Targeting EphA4 might open novel avenues for manipulating such cognitive processes, which are critical for normal memory functions and selective attention (Schmajuk 1997).