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Cell adhesion molecules, such as neuronal cell adhesion molecule (Nr-CAM), mediate cell–cell interactions in both the developing and mature nervous system. Neuronal cell adhesion molecule is believed to play a critical role in cell adhesion and migration, axonal growth, guidance, target recognition and synapse formation. Here, wild-type, heterozygous and Nr-CAM null mice were assessed on a battery of five learning tasks (Lashley maze, odor discrimination, passive avoidance, spatial water maze and fear conditioning) previously developed to characterize the general learning abilities of laboratory mice. Additionally, all animals were tested on 10 measures of sensory/motor function, emotionality and stress reactivity. We report that the Nr-CAM deletion had no impact on four of the learning tasks (fear conditioning, spatial water maze, Lashley maze and odor discrimination). However, Nr-CAM null mice exhibited impaired performance on a task that required animals to suppress movement (passive avoidance). Although Nr-CAM mutants expressed normal levels of general activity and body weights, they did exhibit an increased propensity to enter stressful areas of novel environments (the center of an open field and the lighted side of a dark/light box), exhibited higher sensitivity to pain (hot plate) and were more sensitive to the aversive effects of foot shock (shock-induced freezing). This behavioral phenotype suggests that Nr-CAM does not play a central role in the regulation of general cognitive abilities but may have a critical function in regulating impulsivity and possibly an animal’s susceptibility to drug abuse and addiction.
Cell adhesion molecules (CAMs) mediate cell–cell interactions in both the developing and mature nervous system. Cell adhesion molecules of the immunoglobulin superfamily mediate several aspects of nervous system development, including cell adhesion and migration, axonal growth, fasciculation and guidance, as well as target recognition, synapse formation and plasticity (Grumet 1997; Sakurai et al. 2001).
The elucidation of neuronal cell adhesion molecule (Nr-CAM) expression in mammals has indicated that Nr-CAM may have multiple functions at different locations during nervous system development. Neuronal cell adhesion molecule is expressed in a number of cortical regions, including the hippocampus, olfactory bulb and the corpus callosum (Lustig et al. 2001). In the hippocampus, Nr-CAM is expressed in the major cell layers, including the pyramidal cell layer and the granule cell layer of the dentate gyrus, as well as in the molecular layer (Backer et al. 2002).
Neuronal cell adhesion molecule is ubiquitously expressed in the hippocampus across the life span of mice (Sandi et al. 2005). Given that neural cell adhesion molecules have important functions related to synaptic plasticity (Knafo et al. 2005), and the hippocampus’ and limbic brain regions’ pervasive roles in learning and memory processes, it was of interest to characterize the role of Nr-CAM in regulating cognitive abilities. To that end, here we assess the impact of Nr-CAM on a range of learning tasks (representing multiple cognitive domains), and subsequently, on behaviors indicative of sensory/motor abilities, emotionality and exploration.
In addition to its potential role in learning, recent reports have implicated Nr-CAM in regulating the susceptibility to drug abuse and addiction (Ishiguro et al. 2006), a characteristic that is similar to other Nr-CAMs (Kahn et al. 2005). Specifically, it has been observed that mice with reduced levels of Nr-CAM expression manifest less drug-conditioned place preference, in concordance with the human data that support reduced addiction vulnerability in individuals with similar haplotypes (Hall et al. 2004; Lin et al. 2005). However, it is difficult to interpret this later data given the paucity of information regarding the role of Nr-CAM in learning absent motivational states supported by drugs of abuse. Nonetheless, the suggestion that Nr-CAM contributes to addiction vulnerability and learned drug-related behaviors provided the impetus to assess the role of this adhesion molecule in a wider range of cognitive processes and to more thoroughly characterize the behavioral effects of Nr-CAM deletion in mice.
We have developed a test battery with which to assess the ‘general’ learning abilities of laboratory mice (Kolata et al. 2005, 2007; Matzel et al. 2003, 2006). Here, a similar test battery was used to assess the impact of a brain-wide deletion of the gene for the Nr-CAM. Mice were assessed on five learning tasks (Lashley maze, odor discrimination, passive avoidance, spatial water maze and fear conditioning) and 10 measures of sensory/motor function, emotionality and stress reactivity. These sensory motor tests included measures of pain sensitivity, co-ordination/strength, exploration of novel environments, light/dark preferences, emotionality (e.g. defecation evoked by novel environments or aversive stimulation) and measures of general activity (e.g. running wheel performance).
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Genetic and molecular biological studies have recently linked the Nr-CAM (in both mouse and human populations) with susceptibility to and consequences of addiction (Ishiguro et al. 2006; Minana et al. 2000), aberrant drug-seeking behavior and dysfunctional brain-based drug reward systems (Hitzemann et al. 2003). In support of these contentions, it has been observed that mice with reduced levels of Nr-CAM expression manifest less drug-conditioned place preference (Hall et al. 2004; Lin et al. 2005). However, it was previously unknown whether these deficits in drug conditioning reflect a specific influence of Nr-CAM on drug-motivated behaviors, or whether perturbations of Nr-CAM expression impact learning in a more general manner. Neuronal cell adhesion molecule’s expression in numerous cortical regions, especially the hippocampus, coupled with other Nr-CAM’s involvement in synaptic plasticity (Panicker et al. 2003; Welzl & Stork 2003) suggest that this CAM might be involved more broadly in the acquisition of learned responses. Here, the performance of Nr-CAM knockout mice was compared with wild-type and heterozygous mice on a series of tests of learned and unlearned behaviors. It was determined that brain-wide deletion of Nr-CAM did not affect performance in the majority of our cognitive and sensory/motor tasks. However, behavioral differences in certain specific tasks provide support for the hypothesis that Nr-CAM may play a role in behaviors symptomatic of addiction vulnerability.
Wild-type, heterogeneous and knockout mice did not differ significantly from one another during acquisition in a spatial navigation test (Morris water maze), which depends heavily on hippocampal processes (Bohbot et al. 1996; Deacon et al. 2002). This is revealing as the hippocampus is one of the cortical regions where Nr-CAM is abundantly expressed, and other neuronal CAMs have been implicated in the acquisition of spatial/hippocampal tasks (Cambon et al. 2003; Sandi et al. 2005). The results here suggest that Nr-CAM’s function in the hippocampus is unrelated to performance in tasks that require animals to form a ‘cognitive map’ such as the water maze, and thus distinguishes Nr-CAM from other CAM molecules.
Wild-type, heterogeneous and knockout mice did not differ in their acquisition of learned responses in an egocentric navigation task (Lashley III maze), an odor discrimination task or an associative fear-conditioning task. Because the discrimination task used here is dependent on olfactory abilities, the lack of performance differences related to the expression of Nr-CAM suggests that deletion of this Nr-CAM does not impair olfaction, at least within the limited parameters employed here (Schellinck et al. 2004). Similarly, the lack of impairment in the water maze (which depends on the integration of visual cues) and in fear conditioning (in which animals respond to a noise CS) suggests that at least within the limited range of these tests, visual and auditory abilities were unaffected by Nr-CAM deletion.
Suppression of ongoing behavior in response to a signal (i.e. CS) that predicts impending foot shock is often described as a ‘conditioned emotional response’ (i.e. conditioned ‘fear’; Fanselow & Kim 1994; LeDoux 1997). The lack of an effect of Nr-CAM deletion on fear conditioning is consistent with the absence of group differences in defecation in novel environments, a common measure of unlearned emotionality. In combination, these results suggest that Nr-CAM deletion does not overtly impact emotionality.
Data discussed thus far suggest that brain-wide deletion of Nr-CAM spares forms of learning that are dependent on two components of the limbic system, i.e. the hippocampus (upon which performance in the water maze and odor discrimination tasks rely) and the amygdala [upon which fear conditioning is dependent (Blair et al. 2001; Davis 1992; Phillips & LeDoux 1992)], while the lack of an impairment in odor discrimination consequent to Nr-CAM deletion suggests that fornix function (which is necessary for odor discrimination; Fagan et al. 1985) is spared. Finally, the absence of any impact of Nr-CAM deletion on performance in the Lashley maze is further evidence of normal amygdala and hippocampus function (Dickson & Vanderwolf 1990).
Passive avoidance was the one learning task in the present series of tests where Nr-CAM knockout animals showed impaired performance. Specifically, a brain-wide deletion of Nr-CAM significantly reduced performance of an avoidance response that requires animals to learn to inhibit an overt behavior. Nr-CAM knockout mice did not show an increase in step-down latencies after the step was paired with the onset of aversive stimulation (bright light and siren). This result suggests that Nr-CAM-deficient mice could either not learn the relationship between their behavior and the presentation of the aversive stimulus or that they could not effectively suppress the target behavior. Based on the results of other tests of learning described here, the latter possibility is more parsimonious with available evidence. Furthermore, impaired performance in passive avoidance is not likely attributable to impaired auditory abilities (as necessary to detect the aversive siren used in this task) as mutant animals exhibited a normal (or slightly elevated) auditory startle response and performed normally in a fear-conditioning task in which the danger signal (CS) was white noise.
Mice are highly exploratory (Crawley et al. 1997) and in the passive avoidance task must learn to inhibit this tendency to avoid contact with the aversive event. In at least two measures of exploration (entries into the walled quadrants of the open field and time spent in the lighted side of a dark/light box), Nr-CAM-deficient mice exhibited more exploration, a result that may account for this performance deficit in passive avoidance.
A propensity for drug self-administration has previously been associated with aberrant passive avoidance learning (Seth et al. 2002), and lack of inhibitory control is a hallmark of addiction (Dawe & Loxton 2004). Passive avoidance responses are routinely employed in drug studies to explore the genetic basis of drug vulnerability differences among mice (Bignami 1987; Crawley 2000), and impaired passive avoidance responding has been associated with drug usage (Barrionuevo et al. 2000), prenatal exposure to drugs of abuse (Petkov et al. 1991) and susceptibility to drug usage (Hishida 1996; Sakurai et al. 2001). Notably, impaired passive avoidance responses, like those expressed in Nr-CAM knockout mice, have been observed in rodents with other Nr-CAM deficiencies (Baydas et al. 2005; Cambon et al. 2003; Foley et al. 2000). Data from our other cognitive tasks imply that Nr-CAM knockouts are not impaired (or facilitated) across a wide range of learning domains. However, the impairment in this one task, in combination with the propensity for more exploration in Nr-CAM knockout mice, suggests a specific inability to withhold behavioral responding, not an impairment of general or domain-specific cognitive abilities.
Assessment of sensory/motor performance also produced results suggesting that the lack of Nr-CAM creates a genotype useful in explorations of drug abuse and addiction. In two tests of pain sensitivity and responsiveness, Nr-CAM knockout mice displayed heightened nociception (a decrease in paw lick latencies on a hot plate and prolonged freezing after an unsignaled foot shock) relative to wild-type animals. Increased sensitivity to pain has been implicated in the susceptibility to drug abuse and addiction (Lehofer et al. 1997).
In the open field, Nr-CAM knockout mice spent significantly more time in the unwalled quadrants of the apparatus without an increase in total activity. Exploration of the center quadrants of the open field is often interpreted as indicative of novelty seeking (Stansfield et al. 2004), and novelty seeking is one of the hallmarks of abuse and addiction in human populations and rodent models of abuse (for reviews, see Laviola et al. 2000; Spear 2000). Neuronal cell adhesion molecule knockout’s open-field behavior could be viewed as increased novelty seeking indicative of increased vulnerability to drug abuse type behaviors, as similar open-field behavior has been in other mouse models of addiction (Bowirrat & Oscar-Berman 2005; Gingras & Cools 1997; Stanfield & Trice 1988).
In total, the present results suggest that deletion of Nr-CAM does not promote general deficits in learning. However, Nr-CAM knockout mice performed deficiently in a task that required the suppression of behavior (i.e. passive avoidance). This latter result suggests that these animals may be abnormally impulsive, a result consistent with their propensity to explore stressful areas of a novel open field. In combination with the heightened pain sensitivity exhibited by these animals, these results suggest that Nr-CAM may play a critical function in establishing an animal’s susceptibility to drug abuse and addiction, a speculation supported by recent molecular and genetic work (Ishiguro et al. 2006).