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The metabotropic glutamate receptor subtype 8 (mGlu8) is presynaptically located and regulates the release of the transmitter. Dysfunctions of this mechanism are involved in the pathophysiology of different psychiatric disorders. mGlu8 deficient mice have been previously investigated in a range of studies, but the results are contradictory and there are still many open questions. Therefore, we tested mGlu8-deficient animals in different behavioral tasks that are commonly used in neuropsychiatric research. Our results show a robust contextual fear deficit in mGlu8-deficient mice. Furthermore, novel object recognition, chlordiazepoxide-facilitated extinction of operant conditioning and the acoustic startle response were attenuated by mGlu8 deficiency. We found no changes in sensory processing, locomotor activity, prepulse inhibition, phencyclidine-induced changes in locomotion or prepulse inhibition, operant conditioning, conditioned fear to a discrete cue or in animal models of innate fear and post-traumatic stress disorder. We conclude that mGlu8 might be a potential target for disorders with pathophysiological changes in brain areas where mGlu8 modulates glutamate and gamma-amino butyric acid (GABA) transmission. Our data especially point to anxiety disorders involving exaggerated contextual fear, such as generalized anxiety disorders, and to conditions with disturbed declarative memory.
The metabotropic glutamate receptor subtype 8 (mGlu8) inhibits transmitter release via Gi/o proteins and associated effector pathways (summarized in Kew & Kemp 2005). Like the closely related subtype 7 (mGlu7), mGlu8 is exclusively located on the presynaptic side of glutamatergic and gamma-amino butyric acid (GABA)ergic terminals (Ferraguti et al. 2005; Shigemoto et al. 1997), but has about a 1000 times higher affinity to glutamate than mGlu7 (Schoepp et al. 1999). Because dysfunctions of the glutamatergic and GABAergic brain system are involved in the pathophysiology of different psychiatric disorders (Lewis & Moghaddam 2006; Nemeroff 2003), it has been suggested that mGlu8 might be a potential target for such diseases.
As with antibodies, there is also a lack of specific agonists or antagonists for mGlu8. Therefore, the characterization of mGlu8-deficient mice is currently the only method available to investigate the physiological role of mGlu8. Previous reports state that mGlu8 deficiency is not connected to a very obvious pathological phenotype (e.g. Gerlai et al. 2002). However, when tested in different animal behavioral models, a phenotype was described in innate anxiety models, body weight, pain sensitivity and locomotor activity, but not in animal models of learning, memory, schizophrenia and epilepsy (Duvoisin et al. 2005; Gerlai et al. 2002; Linden et al. 2002; Robbins et al. 2007). It is difficult to compare these studies because different background strains were used, but it is notable that there are contradictory results. For example, mGlu8-deficient animals had an increased body weight in one study (Duvoisin et al. 2005) but not in another (Gerlai et al. 2002). They showed an anxiogenic-like phenotype in several animal models of anxiety (Duvoisin et al. 2005; Linden et al. 2002, 2003; Robbins et al. 2007), but an anxiolytic-like phenotype in a conditioned fear model (Gerlai et al. 2002). Likewise, the phenotype in the open field was described to be hypoactive (Duvoisin et al. 2005), hyperactive (Gerlai et al. 2002) or unchanged (Linden et al. 2002; Robbins et al. 2007). These differences are of critical importance because changes in locomotor activity affect many behavioral tasks.
The aim of the present study was to further analyze the role of mGlu8. Specifically, we were interested in exploring the effects of mGlu8 deficiency in conditioned fear and in learning and memory because the roles of mGlu8 in these phenomena are poorly understood so far. Therefore, we tested mGlu8-deficient mice which are bred on a background (C57BL/6) in animal models of conditioned fear, learning and memory, and also in other models of pivotal importance for neuropsychiatric research.
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- Material and methods
The present study identifies very specific deficits of mGlu8-deficient mice in contextual fear, object recognition, CDP-induced facilitation of extinction and the acoustic startle response; none of which were reported before. At the same time, the behavior of mGlu8-deficient mice in animal models of anxiety and post-traumatic stress disorder (PTSD) (elevated plus and zero maze, light-dark box, fear conditioning/sensitization after traumatic US), in animal models for schizophrenia (PCP-induced hyperactivity and PPI deficits) and in different models of learning (cued fear conditioning, operant learning) was not affected by mGlu8deficiency. The present experiments also showed normal locomotor activity and normal sensory capabilities in mGlu8-deficient mice. Some of these findings stand in contrast to published data.
The latter findings are of great importance, because changes in general locomotor activity complicate the interpretation of behavioral experiments that are dependent on locomotor activity. However, in several independent experiments with different batches of mice, we found no changes in locomotor activity, neither in an open-field experiment focused on detecting such changes nor in the habituation period of the conditioned fear experiments, or of the startle experiments or in the rate of response during operant conditioning. In line with these data, total activity was not affected in the traditional, unconditioned anxiety models, except for one elevated plus-maze session in which mGlu8-deficient mice had an enhanced total number of arm entries. However, this difference was not observed in subsequent sessions, and the total distance traveled was not changed in this experiment. The observation that locomotor activity is not affected by mGlu8 deficiency stands in contrast to studies reporting changes of locomotor activity in mGlu8-deficient mice in both directions (hypoactive: Duvoisin et al. 2005; hyperactive: Gerlai et al. 2002), but supports other studies with similar observations (Linden et al. 2002; Robbins et al. 2007). These discrepancies may arise from the different genetic backgrounds of the mGlu8-deficient animals used (C57BL/6 vs. ICR) or different ages of animals in the studies (between 12 and 24 weeks). Whatever the reason, we can safely conclude that in the experiments in the present study the effects observed are not because of changes in locomotor activity.
In addition, there was no evidence of sensory deficits in mGlu8-deficient mice in the present experiments. The acoustic cue for fear conditioning, as well as the weak acoustic prepulse used in the PPI experiment, had very similar effects in wild-type and mGlu8-deficient mice. Furthermore, different contexts were readily discriminated, and the response to foot shocks was not affected. These findings are in line with previous studies where sensory impairment was only described in the mouse strains used for backcrossing but not in the mGlu8-deficient mice (Duvoisin et al. 2005; Gerlai et al. 2002; Linden et al. 2002; Robbins et al. 2007).
A surprising result of the present study was that mGlu8-deficient mice expressed no anxiogenic phenotype in unconditioned behavioral models of anxiety, including elevated plus maze, elevated zero maze and light-dark box, in contrast to reports in several publications (Duvoisin et al. 2005; Linden et al. 2002, 2003; Robbins et al. 2007), but see also Gerlai et al. 2002. We carried out five independent experiments, all of which showed no effect of genotype. The effect of CDP on operant extinction was partially reversed, but we have shown elsewhere that this effect of CDP is not because of its anxiolytic action (McCabe et al. 2004). It is difficult to explain the discrepancy from the literature with traditional, unconditioned anxiety models. However, one possible explanation is that we always used naive animals in our experiments (except for practical reasons in one light-dark box experiment). This means that we can exclude effects of prior experience in our studies, whereas this is not the case in some of the published experiments (e.g. Duvoisin et al. 2005). It is known that prior test experience affects unconditioned anxiety-like behavior (Carobrez & Bertoglio 2005; Holmes et al. 2001) and this effect could be modulated by mGlu8. Other factors that can influence anxiety behavior are the housing conditions or the laboratory environment (Crabbe et al. 1999; Whitaker et al. 2009). Our animals were housed in environmentally enriched cages which can decrease anxiety behavior (Friske & Gammie 2005). Furthermore, in some earlier studies animals were used which had been backcrossed with ICR mice (Gerlai et al. 2002; Linden et al. 2002, 2003). ICR mice suffer from deficits in vision (Gerlai et al. 2002) and mGlu8 deficiency could potentially increase this deficit. Last, Duvoisin et al. (2005) tested relatively old animals (ca. 6 months) in the published study, whereas we used mice in an age of 2–3 months.
Perhaps more surprising than the lack of anxiogenic-like phenotype in unconditioned tests of anxiety was to find a contextual fear deficit in mGlu8-deficient animals. Conditioned fear tests are believed to be better predictors of clinical efficacy than the tests of innate or unconditioned anxiety (Garakani et al. 2006; Grillon 2002). In several experiments, we found a deficit in the expression of contextual conditioned fear but not of cued fear. The freezing response in the fear conditioning phase was not affected. In a previous study, Gerlai et al. (2002) described a delayed fear response in mGlu8-deficient mice in the fear conditioning phase. However, the maximal freezing responses reported in that study were about 5–15%, indicating a severe deficit in freezing behavior in the ICR strain which was used for backcrossing. C57BL/6 mice (the genetic background of our animals) show a normal freezing response (50–70%).
Our data also showed that the observed contextual fear deficit is a very specific deficit and not indirectly induced. First, a normal freezing response was observed during presentations of the CS showing (1) that the ability to express a freezing response is not affected by mGlu8 deficiency and (2) that there are no general (fear) learning deficits. Second, the ability to discriminate two very similar contexts was not affected in mGlu8-deficient animals, showing that these animals have normal processing of contextual information. However, the contextual fear deficit observed in the present study was not present in animals younger than 2 months or with foot shocks of very high and traumatic intensity. This can be explained by the hypothesis that for the processing of high intensity and traumatic foot shocks other and/or additional neural structures are used (e.g. Walker & Davis 1997), which are not affected by mGlu8 deficiency. In addition, a recently published electrophysiological study indicated that mGlu8 in the hippocampus, a brain region of high importance for contextual fear (Maren 2001), might play different roles during development. It should be noted that mGlu8 is not only densely localized in the hippocampus but also in other brain regions believed to be involved in contextual fear conditioning, such as the amygdala and the septum (Calandreau et al. 2007; Maren 2001).
The present study also detected a mild deficit in novel object recognition, which is considered to be a measure of declarative memory. The mGlu8-deficient mice were able to differentiate the novel objects from the familiar ones, but this effect was much less pronounced than in wild-type animals. This finding is in contrast to Duvoisin et al. (2005) showing no deficits in novel object recognition. However, we used a more complex experimental protocol with four objects, which makes higher demands on memory systems and is probably more sensitive in detecting deficits.
Overall, mGlu8-deficient mice did not express a general learning deficit. The phenotype of mGlu8-deficient animals in fear conditioning to a discrete cue and operant conditioning was normal. However, in that experiment, the facilitation of extinction by the benzodiazepine agonist CDP was also investigated. Our present findings replicate and extend those of previous studies of the effect of CDP (Leslie et al. 2004, 2005; McCabe et al. 2004; Shaw et al. 2004). They show that the effect of CDP in facilitating extinction is further delayed in mGlu8-deficient mice, suggesting that GABAergic processes are diminished in these mice. Metabotropic glutamate receptor subtype 8 is localized on GABAergic terminals (Ferraguti et al. 2005; Shigemoto et al. 1997) and the lack of mGlu8 may lead to an alteration in inhibitory neurotransmission function that is manifest in behavioral changes seen here.
Lastly, we also found a slight reduction in the acoustic startle response, which was not because of a hearing deficit or to lower body weight in mGlu8-deficient animals. However, the startle response is also an indicator of anxiety state (Koch 1999; Koch & Fendt 2003; Lang et al. 1990; Ray et al. 2009; Yilmazer-Hanke et al. 2004). This finding would thus be consistent with an anxiolytic-like phenotype in contextual fear.
Taken together, the present series of experiments detected specific behavioral changes in mGlu8-deficient animals, which point to an important role of mGlu8 in the processing of contextual fear and object recognition. Furthermore, our experiments indicate a role of mGlu8 in the modulation of GABA release. Metabotropic glutamate receptor subtype 8 is located in brain areas processing contextual fear, extinction and object recognition, including the amygdala, hippocampus, the prefrontal and the perirhinal cortex. Both glutamate and GABA as well as the metabotropic glutamate receptors play important roles within these brain sites (Ferraguti et al. 2005; Palazzo et al. 2008; Schmid & Fendt 2006; Winters et al. 2008). Therefore, mGlu8 might be a potential target for disorders with pathophysiological changes in brain areas where mGlu8 modulates glutamate and GABA transmission. The present data especially point to anxiety disorders with extreme contextual fear, such as generalized anxiety disorders (Grillon 2002), and to diseases with disturbed declarative memory.