- Top of page
- Materials and methods
G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels mediate the postsynaptic inhibitory effects of many neurotransmitters and drugs of abuse. The lack of drugs selective for GIRK channels has hindered our ability to study their contributions to behavior. Here, we assessed the impact of GIRK subunit ablation on several behavioral endpoints. Mice were evaluated with respect to open-field motor activity and habituation, anxiety-related behavior, motor co-ordination and ataxia and operant performance. GIRK3 knockout (−/−) mice behaved indistinguishably from wild-type mice in this panel of tests. GIRK1−/− mice and GIRK2−/− mice, however, showed elevated motor activity and delayed habituation to an open field. GIRK2−/− mice, and to a lesser extent GIRK1−/− mice, also displayed reduced anxiety-related behavior in the elevated plus maze. Both GIRK1−/− mice and GIRK2−/− mice displayed marked resistance to the ataxic effects of the GABAB receptor agonist baclofen in the rotarod test. All GIRK−/− mice were able to learn an operant task using food as the reinforcing agent. Within-session progressive ratio scheduling, however, showed elevated lever press behavior in GIRK2−/− mice and, to a lesser extent, in GIRK1−/− mice. Phenotypic differences between mice lacking GIRK1, GIRK2 and GIRK3 correlate well with the known impact of GIRK subunit ablation on neurotransmitter-gated GIRK currents, arguing that most neuronal GIRK channels contain GIRK1 and/or GIRK2. Altogether, our data suggest that GIRK channels make important contribution to a range of behaviors and may represent points of therapeutic intervention in disorders of anxiety, spasticity and reward.
The slow postsynaptic inhibitory effects of many neurotransmitters are mediated by G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels (North 1989). GIRK channels are activated by Gi/o G proteins and are formed by products of four genes (GIRK1–GIRK4) (Mark & Herlitze 2000). Three channel subunits (GIRK1–GIRK3) exhibit broad distributions in the central nervous system, whereas GIRK4 expression is limited (Karschin et al. 1996). The overlapping distribution of neuronal GIRK messenger RNAs suggests the potential for considerable diversity among neuronal GIRK channels (Karschin et al. 1996).
While focused studies have been conducted with all three GIRK−/− lines, a comparative behavioral characterization has not been performed. This type of study was delayed until each line had undergone extensive backcrossing against a well-characterized inbred mouse strain (C57BL/6J). Here, we describe the performance of GIRK−/− mice in established behavioral assays, including open-field activity, rotating rod, elevated plus maze (EPM) and operant tests. Our data highlight the contribution of GIRK channels to a range of behaviors and suggest that therapeutic interventions targeting GIRK channels could be useful in a variety of clinical settings, including anxiety and addiction.
- Top of page
- Materials and methods
We measured the impact of genetic ablation of GIRK1, GIRK2 and GIRK3 in mice using established behavioral paradigms. Assays were chosen that would provide insight into the contribution of GIRK channels to activity, anxiety, muscle co-ordination, ataxia and reward-related behavior. We found that GIRK1−/− mice and GIRK2−/− mice often displayed robust and similar phenotypes, including elevated open-field activity, decreased anxiety-like behavior, decreased baclofen ataxia and increased operant responding for food.
Limited information is available concerning the neurobehavioral profile of GIRK1−/− mice. Like the GIRK2−/− line, GIRK1−/− mice exhibited thermal hyperalgesia and decreased analgesic responses to high doses of intrathecal opioids (Marker et al. 2004). Here, we found that GIRK1−/− mice and GIRK2−/− mice exhibited similar phenotypes with regard to open-field activity and habituation, anxiety and baclofen-induced ataxia. Thus, our work supports the contention that GIRK1 interacts with GIRK2 to form functional channels in most neurons (Karschin et al. 1996; Koyrakh et al. 2005; Liao et al. 1996). Nevertheless, two phenotypes evident in GIRK2−/− mice were attenuated in GIRK1−/− mice. While both lines showed reduced anxiety-related behavior, this phenotype was more pronounced in GIRK2−/− mice. Similarly, GIRK1−/− mice exhibited elevated operant responding for food, but their performance was less pronounced than that of GIRK2−/− mice. There are two likely explanations for these observations. First, some neurons express GIRK2 but not GIRK1, such as the dopamine neurons of the VTA and SNc (Cruz et al. 2004; Eulitz et al. 2007; Inanobe et al. 1999; Karschin et al. 1996). Ventral tegmental area dopamine neurons are vital to reward-related behaviors, including self-administration of food and drugs of abuse (Kalivas & McFarland 2003; Koob 1992; Marinelli et al. 2006; Self 2004; Self & Nestler 1995). Second, although neurotransmitter-induced currents are reduced in neurons from GIRK1−/− mice, the reduction is not always as dramatic as that seen in GIRK2−/− mice (Marker et al. 2006). This is most likely because of the presence of residual current carried by channels formed by GIRK2 and/or GIRK3. Indeed, GIRK2 contains a forward trafficking signal that promotes membrane targeting, while GIRK1 contains an endoplasmic reticulum retention signal and requires coexpression with another GIRK subunit to achieve membrane localization (Hedin et al. 1996; Kennedy et al. 1999; Lunn et al. 2007; Ma et al. 2002).
Behavioral abnormalities were not detected in GIRK3−/− mice in this study. While this may be because of compensatory adaptations that minimize the impact of GIRK3 ablation, some phenotypes have been reported in this mouse line. For example, GIRK3−/− mice exhibited reduced cocaine self-administration (Morgan et al. 2003). In addition, GIRK3−/− mice exhibited thermal hyperalgesia in the hot plate but not the tail flick test (Marker et al. 2002, 2004). Furthermore, the analgesic potency of systemic morphine was reduced in GIRK3−/− mice (Smith et al. in press). Because GIRK3−/− mice showed normal tail flick behavior and responses to intrathecal morphine (Marker et al. 2004), it seems likely that supraspinal GIRK3-containing channels mediate in part the analgesic effect of systemic morphine. Given the otherwise normal behavior of GIRK3−/− mice, these phenotypes suggest the intriguing possibility that GIRK3-containing channels make a selective contribution to the behavioral effects of drugs of abuse.
The mutant mouse lines evaluated in this study were generated using embryonic stem cells from 129-based substrains and were backcrossed against the C57BL/6 strain for 12–22 generations prior to testing. The goal of backcrossing is to minimize inter-subject differences in genetic content that could influence behavioral outcomes. Despite extensive backcrossing, it is not possible to completely eliminate genetic content from the stem cell donor strain using this approach. This is important as differences in the expression level or polymorphisms in ‘hitchhiking’ genes could explain the phenotypic differences between the parent 129 and the C57BL/6 strains and the differences between GIRK−/− mice and wild-type mice. With respect to the paradigms used in this study, behavioral differences have been noted for 129 and C57BL/6 strains. For example, 129 strain shows reduced locomotor activity and heightened anxiety relative to the C57BL/6 strain and a diminished capacity for associative learning (Bothe et al. 2004, 2005; Hagenbuch et al. 2006; Hengemihle et al. 1999; Kelly et al. 2003; Paulus et al. 1999; Rodgers et al. 2002; Tarantino et al. 2000). Thus, contributions of trait-relevant hitchhiking gene(s) in our study are predicted to manifest as hypoactivity, heightened anxiety and diminished operant performance. In contrast, the phenotypes displayed by GIRK−/− mice included heightened locomotor activity, reduced anxiety-related behavior and elevated operant responding for food. Thus, the most parsimonious interpretation of the data is that the lack of the GIRK subunit in question is the dominant source of the phenotypes.
Two aspects of the operant study merit discussion. First, clear genotype-dependent differences in body weights were observed prior to the study. GIRK1−/− and GIRK2−/− mice weighed less than wild-type and GIRK3−/− mice. Thus, the impact of underweight and caloric imbalance on operant performance needs to be considered. Group PR performance was qualitatively similar under both food-restricted and ad libitum conditions, however, arguing that the caloric imbalance cannot fully account for the observed phenotypes. Nevertheless, persistent caloric imbalance (hunger) or elevated metabolism in one or more GIRK−/− lines could impact performance in this test. Certainly, the contribution of GIRK channels to energy homeostasis warrants further examination. Second, the performance of GIRK3−/− mice was consistently lower, although not significantly, than that of wild-type mice in this test. Interestingly, Lüscher and colleagues showed recently that the GIRK channel in VTA dopamine neurons (normally formed by GIRK2 and GIRK3 subunits) from GIRK3−/− mice was more sensitive to GABAB receptor stimulation than GIRK channels in wild-type VTA dopamine neurons (Labouebe et al. 2007). Given that GIRK2 ablation leads to a complete loss of GIRK current in VTA dopamine neurons, it is tempting to speculate that the extent of operant behavior directed at earning food may be inversely proportional to strength of receptor–GIRK channel coupling that exists in VTA dopamine neurons.
Both GIRK1−/− mice and GIRK2−/− mice showed decreased anxiety-like behavior. Given the constitutive and global nature of the GIRK−/− lines, it is not possible to identify the circuit(s) or neurotransmitter system(s) that explain the anxiolytic phenotype. Nevertheless, the elevated anxiety-related behaviors seen in GABAB(1) receptor knockout mice argue that the loss of GABAB–GIRK signaling is an unlikely explanation for the anxiolytic phenotype in GIRK−/− mice (Mombereau et al. 2004). Although other explanations are tenable, our observations are more consistent with those of a recent study showing that chronic administration of the selective serotonin reuptake inhibitor fluoxetine exerts a beneficial influence on a rodent model of depression through suppression of GIRK-dependent signaling in the dorsal raphé nucleus (Cornelisse et al. 2007). Acute inhibition of GIRK channels by fluoxetine was also reported in other systems (Takahashi et al. 2006). Thus, the potential contribution of GIRK channels to emotional disorders and the actions of a common class of antidepressant compounds warrant additional attention.
In clinical settings, baclofen is used to treat spasticity and muscular rigidity associated with cerebral palsy, muscular sclerosis, amyotrophic lateral sclerosis and spinal cord injury (Brogden et al. 1974; Vacher & Bettler 2003). Baclofen also exerts potent antinociceptive effects at the spinal level (Slonimski et al. 2004). Side-effects of baclofen are pronounced, however, and include sedation, hypothermia, amnesia, dizziness, headache, insomnia and sexual dysfunction (Denys et al. 1998; Kofler et al. 2002). These and other unfavorable properties limit its use in clinical medicine and behavioral pharmacology (Vacher & Bettler 2003). Here, we show that GIRK channels, likely formed by GIRK1 and GIRK2, make a significant contribution to baclofen ataxia. Thus, it is worth considering the possibility that drugs that directly activate GIRK channels may evoke the beneficial effects of baclofen (e.g. analgesia and muscle relaxation) while showing diminished off-target effects.
In summary, our findings implicate GIRK channels in motor activity, anxiety, reward and baclofen ataxia. The channels involved in these behaviors are likely formed by GIRK1 and/or GIRK2. Future studies will strive for increased resolution with regard to GIRK modulation. The site-specific injection of drugs and/or genetic reagents that perturb GIRK-dependent signaling with molecular and spatial precision in wild-type mice will be quite informative and will obviate concerns linked to constitutive gene ablation.