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Neuropeptide Y (NPY) acting through Y1 receptors reduces anxiety- and depression-like behavior in rodents, whereas Y2 receptor stimulation has the opposite effect. This study addressed the implication of Y4 receptors in emotional behavior by comparing female germ line Y4 knockout (Y4−/−) mice with control and germ line Y2−/− animals. Anxiety- and depression-like behavior was assessed with the open field (OF), elevated plus maze (EPM), stress-induced hyperthermia (SIH) and tail suspension tests (TST), respectively. Learning and memory were evaluated with the object recognition test (ORT). In the OF and EPM, both Y4−/− and Y2−/− mice exhibited reduced anxiety-related behavior and enhanced locomotor activity relative to control animals. Locomotor activity in a familiar environment was unchanged in Y4−/− but reduced in Y2−/− mice. The basal rectal temperature exhibited diurnal and genotype-related alterations. Control mice had temperature minima at noon and midnight, whereas Y4−/− and Y2−/− mice displayed only one temperature minimum at noon. The magnitude of SIH was related to time of the day and genotype in a complex manner. In the TST, the duration of immobility was significantly shorter in Y4−/− and Y2−/− mice than in controls. Object memory 6 h after initial exposure to the ORT was impaired in Y2−/− but not in Y4−/− mice, relative to control mice. These results show that genetic deletion of Y4 receptors, like that of Y2 receptors, reduces anxiety-like and depression-related behavior. Unlike Y2 receptor knockout, Y4 receptor knockout does not impair object memory. We propose that Y4 receptors play an important role in the regulation of behavioral homeostasis.
There is evidence that both Y1 and Y2 receptors are relevant to emotional behavior. Intracerebroventricular injection of NPY reduces anxiety- and depression-related behavior in several animal models, this action being primarily mediated by Y1 receptors (Heilig 2004; Kask et al. 2002; Primeaux et al. 2005; Redrobe et al. 2002). Neuropeptide Y acting through Y2 receptors enhances anxiety- and depression-like behavior as deduced from the behavioral characterization of Y2 receptor knockout (Y2−/−) mice (Redrobe et al. 2003; Tschenett et al. 2003). In addition, Y2 receptors are relevant to cognitive functions, given that Y2−/− mice exhibit impaired performance in the Morris water maze and object recognition tests (ORT) (Redrobe et al. 2004b).
The possible role of Y4 receptors in the control of affective behavior has not yet been examined. Albeit less widely distributed in the brain than Y1 and Y2 receptors, the presence of Y4 receptors in hypothalamus, limbic system and medullary brainstem (Dumont et al. 1998; Fetissov et al. 2004; Heilig 2004; Kask et al. 2002; Parker & Herzog, 1999; Stanic et al. 2006) is consistent with a putative role of Y4 receptors in emotional and stress-related behavior. As Y4 receptor-selective antagonists are not yet available, the first and major aim of the present study was to evaluate anxiety-like and depression-related behavior in Y4 receptor knockout (Y4−/−) mice. Anxiety-related behavior was assessed with the open field (OF), elevated plus maze (EPM) and stress-induced hyperthermia (SIH) tests, while depression-related behavior was evaluated with the tail suspension test (TST). Locomotor activity in the novel and familiar environment of the home cage was also evaluated.
As Y2−/− mice have a deficit in learning and memory (Redrobe et al. 2004b), the second aim was to test Y4−/− mice for their performance in the ORT and to compare them with Y2−/− and control mice.
The presence of NPY and Y4 receptors in the hypothalamus led us to ask whether NPY acting through Y4 receptors has an impact on the hypothalamic–pituitary–adrenal (HPA) axis, which is involved in the regulation of depression-related behavior (Holsboer 2000). The aim of the third study was hence to determine the plasma levels of corticosterone at baseline and following exposure to restraint stress in order to obtain an index of HPA axis activity in control and Y4−/− mice.
As SIH test, TST and the corticosterone response test have not yet been performed with Y2−/− mice, the aim of the fourth study was to compare control, Y2−/− and Y4−/− mice in their performance in these tests.
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The current data show that, relative to control animals, Y4−/− mice exhibit reduced anxiety-like and depression-related behavior on the OF and EPM and in the TST, respectively. These effects of Y4 receptor deletion resemble those of Y2 receptor knockout (Redrobe et al. 2003; Tschenett et al. 2003) and Y2 receptor blockade (Bacchi et al. 2006). In contrast, Y4−/− and Y2−/− mice differ in their cognitive behavior, given that Y4−/− mice perform as well as control animals, whereas Y2−/− mice have a deficit in object memory as shown before (Redrobe et al. 2004b).
Relative to control animals, Y4−/− and Y2−/− mice exhibited diminished anxiety-related behavior as assessed in the EPM and OF tests. Knockout of either the Y4 or Y2 receptor gene increased the time spent in the central area of the OF and on the open arms of the EPM. Overall locomotor activity as assessed by the total traveling distance on the EPM was also enhanced in Y4−/− and Y2−/− mice, whereas in the OF test, only Y4−/− mice traveled a significantly longer distance than the control mice. Although the magnitude of anxiety-related behavior in the EPM and OF tests can be influenced by locomotion (File 2001), we conclude that the anxiolytic effect of Y4 and Y2 receptor deletion is not directly related to increased locomotor activity for a number of reasons. First, Y4 and Y2 receptor gene knockout was associated with a selective increase in open arm entry and open arm time on the EPM, while the respective parameters for the closed arms were diminished. Enhanced locomotor activity in male Y2−/− mice was noted in the OF but not on the EPM (Redrobe et al. 2003; Tschenett et al. 2003). Second, the test-dependent increase in locomotor activity in Y2−/− mice is conceivably related to the decrease in visual attention and increase in impulsivity caused by Y2 receptor knockout (Greco & Carli 2006). Third, the increased locomotion of Y4−/− mice on the OF and EPM seems to be related to the novelty of the test environment because a similar increase in locomotion was seen when the animals were put into a novel home cage, whereas in a familiar home cage, locomotion was unchanged in Y4−/− mice and even decreased in Y2−/− mice.
Most experimental studies of emotional behavior are performed with male rather than female rodents (Palanza 2001). If seen as a model for human disease, this experimental approach is at variance with epidemiological evidence that anxiety and mood disorders have a higher prevalence in women than in men (Gorman, 2006; Palanza 2001; Simonds & Whiffen, 2003). For this reason, we decided to study female mice and to explore the role of Y4 and Y2 receptors in the emotional behavior of this gender. Although the estrus cycle was not determined, we consider it unlikely that our data were biased by this potentially confounding factor. Thus, the experiments were performed in the strict absence of male mice, and the coefficient of variation for the EPM data in female control and Y2−/− mice was not greater than that in male mice of identical genetic background (Tschenett et al. 2003). Furthermore, the behavior of mice on the EPM does not vary significantly with the different phases of the estrus cycle that is synchronized not only among cage mates but also across cages (Painsipp et al. 2007). Fourth, male Y4−/− mice have the same anxiolytic-like and antidepressant-like phenotype as female Y4−/− mice (G. Sperk, personal communication).
Neuropeptide Y acting through Y1 receptors has been involved in the circadian control of homeostatic functions such as motor activity, exploration and anxiety-related behavior (Karl et al. 2006; Yannielli & Harrington 2001). We have found here that knockout of either the Y4 receptor or the Y2 receptor has an impact on the diurnal fluctuation of baseline rectal temperature (T1). The high value of T1 in Y4−/− and Y2−/− mice throughout the scotophase is conceivably related to the enhanced intake of water during that period (Wultsch et al. 2006). In keeping with previous data (Sainsbury et al. 2002a,c), our results indicate that the circadian regulation of body temperature and energy homeostasis is altered in Y4−/− and Y2−/− mice, and it awaits to be elucidated which mechanisms (e.g. motor activity, water and food intake) account for the changes in the diurnal T1 profile.
Relative to the EPM test, the SIH test has the advantage of assessing anxiety in a locomotion-independent manner. In the current study, however, this test was complicated by the circadian and genotype-related alterations in T1 and the interaction between these factors. Stress-induced hyperthermia (ΔT) is thought to be a homeostatic reaction that involves the central as well as sympathetic nervous system (DiMicco et al. 2006; Liu et al. 2003; Oka et al. 2001) and depends on light conditions and day time (Peloso et al. 2002). The present study showed that ΔT in control mice was maximal at noon and midnight when T1 was lowest. In Y4−/− and Y2−/− mice, SIH was practically absent during the dark phase when T1 was highest. It is very likely, therefore, that SIH in Y4−/− and Y2−/− mice during the scotophase has been cut short by a ceiling effect. As a consequence, the SIH test cannot be used to assess anxiety if T1 is changed by the experimental manipulation under study (Painsipp et al. 2007).
In the TST, the immobility time of Y4−/− mice was shortened, which is thought to reflect a reduction of depression-like behavior (Cryan et al. 2005). A similar observation in female Y2−/− mice is consistent with a previous report that male Y2−/− mice spend less time immobile in the forced swim test than the control animals (Tschenett et al. 2003).
The deficit of male Y2−/− mice in novel object recognition and object memory (Redrobe et al. 2004b) has been confirmed here with female Y2−/− mice. As Y4−/− mice failed to display a similar cognitive impairment, Y4 receptors do not seem to play a significant role in nonspatial working memory, which the ORT is thought to evaluate (Dodart et al. 1997; Ennaceur & Delacour 1988; Redrobe et al. 2004b). The cognitive deficits associated with Y2 receptor knockout are consistent with the region-specific effects of intracerebral NPY injections and the amnesia resulting from NPY overexpression in the hippocampus (Flood et al. 1989; Redrobe et al. 2004a; Thorsell et al. 2000). A more complete analysis of cognition in Y4−/− mice was beyond the scope of this study.
Compared with Y2 receptors, Y4 receptors are less abundant in the brain, and their functional implications are little understood because of a lack of selective Y4 receptor antagonists. Although PP, the preferential agonist at Y4 receptors, is largely absent from the brain, Y4 receptors have been localized to the medial and basolateral amygdala, ventral tegmental area, hippocampus, hypothalamus, locus coeruleus and medulla of the rodent brain (Campbell et al. 2003; Dumont et al. 1998; Fetissov et al. 2004; Parker & Herzog 1999). In the hypothalamus, Y4 receptors are involved in presynaptic inhibition of transmitter release (Acuna-Goycolea et al. 2005), a mechanism that could explain why Y4 receptor knockout results in similar alterations of emotional behavior as Y2 receptor deletion. The anxiolytic-like phenotype of Y4−/− mice is consistent with the anxiogenic phenotype of PP-overexpressing mice (Ueno et al. 2007). As intracerebroventricular PP fails to alter anxiety-related behavior (Asakawa et al. 1999) while chronic peripheral administration of PP reduces anxiety (Asakawa et al. 2003), it is conceivable that PP modifies anxiety- and depression-like behavior through an action in the periphery or in the area postrema outside the blood–brain barrier (Dumont et al. 2007; Larsen & Kristensen 1997). In this context, it is worth mentioning that both Y2−/− and Y4−/− mice exhibit increased levels of circulating PP (Sainsbury et al. 2002a,c).
In conclusion, our data show that deletion of Y4 receptors, like that of Y2 receptors, reduces anxiety- and depression-related behavior. Although developmental compensations in germ line gene knockout mice may be a confounding factor, our data indicate that, if such adaptations occurred, they were insufficient to balance the deficit in Y4 and Y2 receptors, respectively. This instance attests to a novel and important role of Y4 receptors in the control of emotional behavior and diurnal homeostasis and warrants further examination of Y4 receptor function at the cellular level and exploration of Y4 receptors as a novel drug target.