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Keywords:

  • Anxiety-related behavior;
  • depression-related behavior;
  • elevated plus maze test;
  • home-cage activity;
  • neuropeptide Y;
  • object recognition;
  • open-field test;
  • tail suspension test;
  • Y2 receptors;
  • Y4 receptors

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

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.

Neuropeptide Y (NPY) is widely distributed in the central nervous system where it is involved, among others, in the homeostatic regulation of mood, anxiety, stress sensitivity and cognition (Harro 2006; Heilig 2004; Karl & Herzog 2007; Kask et al. 2002; Lin et al. 2004). Its physiological actions are mediated by several classes of NPY receptors, five of which (Y1,Y2,Y4,Y5 and Y6) have been elucidated at the gene and protein level (Michel et al. 1998; Redrobe et al. 2004a). Coupled to Gi/o signaling pathways, these Y receptors mediate the functional implications of NPY in the brain.

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.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Experimental animals

This study was carried out with adult female mice, weighing 21–33 g, that were housed in groups of three to four per cage under controlled temperature (21°C) and a 12-h light/dark cycle (lights on at 0600 h and lights off at 1800 h). All experiments were approved by an ethical committee at the Federal Ministry of Science and Research of the Republic of Austria and conducted according to the Directive of the European Communities Council of 24 November 1986 (86/609/EEC). The experiments were designed in such a way that the number of animals used and their suffering were minimized.

Specifically, the experiments were performed with germ line Y2−/− and Y4−/− mice and non-induced conditional Y2 and Y4 receptor knockout (FY2 and FY4) mice, which were bred in the Department of Pharmacology of the Medical University of Innsbruck (Innsbruck, Austria), while all experiments were carried out at the Medical University of Graz. The generation of Y2−/−, Y4−/−, FY2 and FY4 mice has been described previously (Sainsbury et al. 2002a,c). Germ line Y2−/− and Y4−/− mice were generated from the same founders on the same mixed C57BL/6:129/SvJ (50%:50%) background as the conditional FY2 and FY4 knockout mice. Germ line Y2−/− and Y4−/− mice were obtained by crossing chimeric mice carrying a Y2 floxed gene (Y2lox/lox) or a Y4 floxed gene (Y4lox/lox), respectively, with oocyte-specific Cre recombinase-expressing C57BL/6 mice (Sainsbury et al. 2002a,c). Non-induced conditional FY2 and FY4 knockout mice were used as controls in all experiments and termed control mice throughout the paper. As shown before, these non-induced conditional Y2lox/lox and Y4lox/lox mice do not differ from wild-type mice as the level of expression of Y2 and Y4 receptors is not influenced by the introduction of the loxP sites (Sainsbury et al. 2002a,c). The deletion or presence of Y2 and Y4 receptors in the germ line and non-induced conditional knockout mice was verified by receptor autoradiography using [125I]PYY3–36 and [125I]PP, respectively, in situ hybridization (data not shown) as well as by polymerase chain reaction using oligonucleotide primers recognizing DNA sequences adjacent to the loxP sites flanking the deleted or residing Y2 and Y4 receptor genes (Sainsbury et al. 2002a,c).

As reported previously (Sainsbury et al. 2002a,c; Redrobe et al. 2003; Tschenett et al. 2003), the knockout animals did not have any gross abnormalities, did not exhibit any obvious signs of sensory deficits and appeared healthy. There was no significant difference in the body weight between the different genotypes used in this study.

Experimental protocols

Four studies with three different cohorts of animals of each genotype were performed. In the first study, the mice were subjected to a sequence of three behavioral tests spaced apart for at least 1 week. The series of behavioral tests was started with the EPM test, continued with the TST and completed with the SIH test. This series of behavioral tests was replicated with a second group of animals, and as the results of the two test series were very similar, the data were pooled and are presented as one data set. In the second study, the effect of stress on the levels of circulating corticosterone was examined in a separate group of mice of each genotype under study. To this end, the levels of circulating corticosterone were measured in the absence of stress and following a 30-min exposure to moderate restraint stress. In the third study, the mice were subjected to the OF test followed by the ORT 1 week later. The fourth study was carried out to measure locomotor activity in the home cage at the time window during which the TST as well as the EPM and OF tests were performed.

Behavioral tests

Prior to all behavioral tests, the mice were allowed to adapt to the test room (22 ± 1°C, 50 ± 15% relative air humidity, lights on at 0600 h, lights off at 1800 h and maximal light intensity of 100 lux) for at least 2 days.

Home-cage activity

The locomotor activity of mice in the home cage was recorded with a six-cage LabMaster system (TSE Systems, Bad Homburg, Germany). Each cage was fitted with a photobeam-based activity monitoring system that recorded every ambulatory movement (Theander-Carrillo et al. 2006). Locomotion was evaluated for the time window (1000–1400 h) during which the TST, EPM and OF tests were carried out. Locomotor activity during this time window was recorded twice. The first recording was made immediately after the animals had been placed for the first time in the home cage, while the second recording was taken 2 days later when the mice had become familiar with the home cage.

Open-field test

The OF consisted of a box (50 × 50 × 30 cm) that was made of opaque gray plastic and illuminated by 80 lux at floor level. The ground area of the box was divided into a 36 × 36 cm central area and the surrounding border zone. Mice were individually placed in a corner of the OF, and their behavior during a 5-min test period was tracked by a video camera positioned above the center of the OF and recorded with the software VideoMot2 (version 5.73; TSE Systems). This software was used to evaluate the time spent in the central area, the number of entries into the central area and the total distance traveled in the central area as well as in the whole OF. The OF test was carried out between 1000 and 1400 h.

Elevated plus maze test

The animals were placed in the center of a maze with four arms arranged in the shape of a plus (Belzung & Griebel 2001; Pellow & File 1986). The maze consisted of a central quadrangle (5 × 5 cm), two opposing open arms (30 cm long, 5 cm wide) and two opposing closed arms of the same size but equipped with 15 cm high walls at their sides and the far end. The device was made of opaque gray plastic and elevated 70 cm above the floor. The light intensity at the central quadrangle was 70 lux, on the open arms 80 lux and in the closed arms 40 lux.

At the beginning of each trial, the animals were placed on the central quadrangle facing an open arm. The movements of the animals during a 5-min test period were tracked by a video camera above the center of the maze and recorded with the software VideoMot2 (TSE Systems). This software was used to evaluate the animal tracks and to determine the number of their entries into the open and closed arms, the time spent on the open and closed arms and the total distance traveled in the open and closed arms during the test session. Entry into an arm was defined as the instance when the mouse placed its four paws on that arm. The EPM test was carried out between 1000 and 1400 h.

Stress-induced hyperthermia test

Measurement of the basal temperature in mice with a rectal probe represents a stressor that causes an increase in the temperature by about 1–1.5°C within 15 min (Olivier et al. 2003; Zethof et al. 1994). Measurement of the basal temperature (T1) was followed by a second measurement of the temperature (T2) 13 min later. This time interval had been found in pilot experiments to best portray the maximal increase in temperature that returned to baseline levels within the following hour. Being determined with a digital thermometer (BAT-12; Physitemp Instruments, Clifton, NJ, USA) equipped with a rectal probe for mice, the SIH was calculated as the difference ΔT = T2 − T1. As SIH depends on both the time of the day and the light conditions (Peloso et al. 2002), the SIH test was carried out at four time slots within a 24-h cycle starting at 0700 h in the morning. The same mice were consecutively tested at all four time slots. The tests at 0700–0730 h and at 1300–1330 h were performed at a light intensity of 100 lux. The following two tests were conducted at 1900–1930 h and 0100–0130 h at red light conditions, i.e. in complete darkness for the rodents.

Tail suspension test

Following exposure to the inescapable stress of being suspended by their tail, mice first struggle to escape but sooner or later attain a posture of immobility (Cryan et al. 2005; Liu & Gershenfeld 2001; Steru et al. 1985). Mice were suspended by their tail with a 1.9-cm wide strapping tape (Omnitape®; Paul Hartmann AG, Heidenheim, Germany) to the lever of a force displacement transducer (K30 type 351; Hugo Sachs Elektronik, Freiburg, Germany) that was connected to a bridge amplifier (type 301; Hugo Sachs Elektronik). The force displacement signals caused by the struggling animal were fed, through an A/D converter (PCI-AD16LC; Kolter Electronic, Erftstadt, Germany), into a personal computer and evaluated with custom-made software. The sampling frequency was 20 Hz. Each trial took 6 min and was carried out at a light intensity of 20 lux. The total duration of immobility was calculated as the time during which the force of the animals’ movements was below a preset threshold. This threshold was determined to be ±7% of the animal’s body weight, and immobility was assumed when at least four digits recorded in continuity (equivalent to a time of 0.2 seconds) were within this threshold range. The validity of the threshold parameters was proved by a highly significant (P < 0.001) Pearson correlation coefficient (r = 0.641) between the software output data and the duration of immobility recorded with a stopwatch in 22 animals. The TST was carried out between 1000 and 1400 h.

Object recognition test

The ORT was performed in the OF box (50 × 50 × 30 cm) that was made of opaque gray plastic and illuminated by 80 lux at floor level. The objects to be discriminated were a rough metal tube (outer diameter 3.1 cm, inner diameter 1.9 cm, length 3.8 cm) and a rough Teflon column (3.2 × 2.9 × 5.8 cm, length × width × height) with a hole (diameter 1.3 cm) at half-height. After each trial, the objects and the OF were cleaned with ethanol (96%) to eliminate olfactory cues (Dodart et al. 1997). Mice were habituated to the OF for 20 min each day during four consecutive days, this period being followed by a pause of 4 days before the ORT. On the test day, two identical objects were placed on the centerline of the OF, 9 cm from each box end (Redrobe et al. 2004b). The animals were allowed to explore the two objects for 5 min during which the exploratory activity directed at each object was tracked by a video camera above the center of the OF and recorded with the software VideoMot2 (TSE Systems). After a delay of 6 h, the animals were re-exposed to one familiar object together with a novel object not used in the acquisition phase, and the exploratory behavior directed at each object recorded during another 5-min test period (Redrobe et al. 2004b). The position of each object was alternated between the trials, and the object chosen to be familiar and novel was changed from mouse to mouse. The performance of each mouse was expressed by the memory index (MI) that was calculated according to the formula MI = (tn − to)/(tn + to), where to represents the time exploring the familiar object and tn represents the time exploring the novel object (Redrobe et al. 2004b).

Circulating corticosterone

The plasma levels of corticosterone were determined between 1200 and 1400 h, both at baseline and following exposure to stress. Baseline levels of circulating corticosterone were measured in animals that stayed in their home cage undisturbed until the time of trunk blood collection. For exposure to moderate restraint stress at room temperature, the mice were placed in a tube of 3 cm diameter and 11 cm length, either end of which had an opening of 0.5 cm diameter to permit exchange of air. After a period of 30 min restraint, the animals were returned to their home cage for a period of 30 min. At the end of this period, the animals were deeply anesthetized with pentobarbital (100 mg/kg intraperitoneally) and decapitated within 3 min of the pentobarbital injection. The same procedure was used to collect blood for determination of baseline corticosterone levels. Trunk blood was collected into vials coated with ethylenediamine tetraacetate (Greiner, Kremsmünster, Austria) kept on ice. Following centrifugation for 20 min at 4°C and 1200 g, blood plasma was collected and stored at −20°C until assay. The plasma levels of corticosterone were determined with an enzyme immunoassay kit (Assay Designs, Ann Arbor, MI, USA). According to the manufacturer’s specifications, the sensitivity of the assay was 27 pg/ml, and the intra-assay and interassay coefficient of variation amounted to 7.7% and 9.7%, respectively.

Statistics

Statistical evaluation of the results was performed on SPSS 14.0 (SPSS Inc., Chicago, IL, USA). One-way, two-way or three-way analysis of variance (anova), for single or repeated measurements, was used to dissect statistical differences for the factors genotype and, if applicable, time and/or treatment. In case of sphericity violations, the Greenhouse–Geisser correction was applied. The homogeneity of variance was analyzed with the Levene test. Post hoc analysis of group differences was performed with the Tukey’s HSD (honestly significant difference) test when the variances were homogeneous and with the Games–Howell test when the variances were unequal. Probability values of <0.05 were regarded as statistically significant. All data are presented as means ± SEM, n referring to the number of mice in each group.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Home-cage activity

Home-cage activity was recorded during the photophase from 1000 to 1400 h. Locomotor activity recorded immediately after the animals had been placed in the home cage on day 1 (Fig. 1a,c) was considerably higher than that on day 3 (Fig. 1b,d) when the mice had become familiar with their environment. This difference was seen for control, Y4−/− and Y2−/− mice irrespectively of whether a 1-h period (Fig. 1a,b) or a 4-h period (Fig. 1c,d) was analyzed. Analysis of variance showed genotype-related differences in locomotion on day 1 (1000–1100 h: F2,29 = 8.82, P = 0.001; 1000–1400 h: F2,29 = 10.55, P < 0.001) and day 3 (1000–1100 h: F2,29 = 2.16, P = 0.13; 1000–1400 h: F2,29 = 19.74, P < 0.001). Post hoc analysis indicated that in the non-familiar environment, Y4−/− mice but not Y2−/− mice displayed higher locomotor activity than the control mice (Fig. 1a,c), whereas in the familiar environment, Y4−/− mice did not differ from controls and Y2−/− mice moved less than the control animals (Fig. 1b,d).

image

Figure 1. Locomotor activity in the home cage recorded in control, Y4−/− and Y2−/− mice between 1000 and 1400 h. Two recordings were taken from each mouse: immediately after the animals had been placed in the home cage on day 1 (a, c) and 2 days later (day 3) when the mice had become familiar with the home cage (b, d). The data show the counts of photobeam crossings accumulated for periods of 1 h (a, b) and 4 h (c, d). The values represent means ± SEM, n as indicated in brackets. **P < 0.01 versus control mice.

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Open-field test

The OF test was first used to examine the locomotor/exploratory and anxiety-related behavior of control, Y4−/− and Y2−/− mice (Fig. 2). The time spent in the central area and the number of entries into the central area were considered to be indices of anxiety and expressed as percentage of the total test duration and of the total number of entries into any zone during the whole test session, respectively. Analysis of variance showed a genotype-related difference in the time spent in the central area (F2,23 = 4.585, P = 0.02), and post hoc analysis showed that Y4−/− and Y2−/− mice spent significantly more time in the central area than the control mice (Fig. 2a). A similar observation was made with regard to the number of central area entries that exhibited a genotype-related difference (F2,23 = 10.043, P < 0.001), given that Y4−/− but not Y2−/− mice entered the central area significantly more often than the control animals (Fig. 2b).

image

Figure 2. Behavior of Y4−/−, Y2−/− and control mice in the OF test. The graphs show the time spent in the central area (a), the number of entries into the central area (b) and the total distance traveled (c) during the 5-min test session. The time spent in the central area is expressed as percentage of the total test duration, and the number of entries into the central area is given as percentage of the total number of entries into any zone during the whole test session. The values represent means ± SEM, n as indicated in brackets. *P < 0.05, **P < 0.01 versus control mice.

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Further analysis showed that knockout of the Y4 receptor gene caused an increase in the locomotor/exploratory activity in the OF (Fig. 2c). Thus, the total traveling distance in the OF (F2,23 = 16.598, P < 0.001) during the test session differed between the genotypes, and post hoc analysis showed that Y4−/− but not Y2−/− mice exhibited greater locomotor activity than the control mice (Fig. 2c).

Elevated plus maze test

The anxiety-related behavior of control, Y4−/− and Y2−/− mice was further assessed with the EPM test (Fig. 3a–f) in which the time spent on the open arms and the number of entries into the open arms were taken as established indices of anxiety. These parameters were expressed as percentage of the total time spent on any arm and of the total number of entries into any arm during the 5-min test session. Analysis of variance showed a genotype-related difference in the time spent on the open arms (F2,38 = 6.07, P < 0.01). Post hoc analysis showed that both Y4−/− and Y2−/− mice spent significantly more time on the open arms than the control mice (Fig. 3a). This result was reproduced by the number of open arm entries that exhibited a genotype-related difference (F2,38 = 6.58, P < 0.01), given that both Y4−/− and Y2−/− mice entered the open arms significantly more often than the control animals (Fig. 3b). In contrast, the number of entries into the closed arms was significantly smaller (F2,38 = 6.58, P < 0.01) in Y4−/− and Y2−/− mice than in the control mice (Fig. 3d). Likewise, the time spent on the closed arms was significantly (F2,38 = 3.88, P < 0.05) shortened in Y4−/− mice but insignificantly (P = 0.11) reduced in Y2−/− mice (Fig. 3c).

image

Figure 3. Behavior of Y4−/−, Y2−/− and control mice in the EPM test. The graphs show the time spent on the open arms (a), the number of entries into the open arms (b), the time spent on the closed arms (c), the number of entries into the closed arms (d), the total distance traveled in the open and closed arms (e) and the total number of entries into any arm (f) during the 5-min test session. The time spent on the open or closed arms is expressed as percentage of the total time spent on any arm, and the number of entries into the open or closed arms is given as percentage of the total number of entries into any arm during the 5-min test session. The values represent means ± SEM, n as indicated in brackets. *P < 0.05, **P < 0.01 versus control mice.

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In order to assess locomotor activity on the EPM, the total distance traveled in the open and closed arms and the total number of entries into any arm during the 5-min test session was analyzed. Both the total traveling distance (F2,38 = 12.27, P < 0.001) and the number of total arm entries (F2,38 = 9.84, P < 0.001) differed between the genotypes, and post hoc analysis showed that both Y4−/− and Y2−/− mice exhibited greater locomotor activity than the control mice (Fig. 3e,f).

Stress-induced hyperthermia test

The SIH test was carried out at four time slots within a 24-h cycle, i.e. at 0700–0730 h and 1300–1330 h at 100 lux as well as at 1900–1930 h and 0100–0130 h under red light conditions. The baseline rectal temperature (T1) of control mice showed characteristic diurnal fluctuations (Fig. 4a), with minima at 1300–1330 h and 0100–0130 h and maxima at 0700–0730 h and 1900–1930 h, i.e. following the change in the light conditions (lights off at 1800 h and lights on at 0600 h). Analysis of variance showed that T1 differed with regard to both the time of the day (F3.73,89.60 = 39.81, P < 0.001) and the genotype (F(2,24) = 6.95, P < 0.01). In addition, there was a significant interaction between the factors genotype and time of the day (F7.47,89.60 = 12.32, P < 0.001). The circadian oscillations in T1 were flattened in Y2−/− mice in which only one minimum at 1300 h was discernible; in addition, the T1 of Y2−/− animals at 0100–0130 h was significantly (P < 0.001) higher than that of the control animals (Fig. 4b). Y4−/− mice had a lower T1 at 0700–0730 h and 1300–1330 h and a higher T1 at 0100–0130 h (P < 0.001) than the control mice (Fig. 4c).

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Figure 4. Behavior of control, Y2−/− and Y4−/− mice in the SIH test in which the rectal temperature was measured twice at an interval of 13 min. In the graphs on the left-hand side (a–c), the black columns depict the rectal temperature recorded at the first measurement (T1) and the white columns show the rectal temperature recorded at the second measurement (T2). The graphs on the right-hand side (d–f) show the SIH (ΔT = T2 − T1). The values represent means ± SEM, n as indicated in brackets. *P < 0.05 and **P < 0.01 refer to significant differences in T1 and ΔT versus control mice.

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Stress-induced hyperthermia was determined by a second measurement of rectal temperature (T2) 13 min after recording of T1 and expressed as the difference ΔT = T2 − T1. At this second measurement, the rectal temperature (T2) did not exhibit any genotype-related difference, but there was a significant interaction between the factors genotype and time of the day (F7.47,89.60 = 12.32, P < 0.001). Thus, T2 varied with the time of the day (F3.73,89.60 = 39.81, P < 0.001) and exhibited a minimum at 1300–1330 h (Fig. 4a–c). Analysis of variance of ΔT (SIH) showed that this parameter depended both on the time of the day (F3,96 = 23.76, P < 0.01) and on the genotype (F2,96 = 32.64, P < 0.01) and that there was a significant interaction between the factors genotype and time of the day (F6,96 = 10.44, P < 0.01). In control animals, ΔT showed characteristic diurnal fluctuations (Fig. 4d), with minima at 0700–0730 h and 1900–1930 h and maxima at 1300–1330 h and 0100–0130 h. ΔT in Y2−/− mice did not differ from that in control mice, except at the time slot of 0100–0130 h when there was no longer any SIH, and the negative ΔT was significantly smaller (P < 0.01) than in control mice (Fig. 4e). Y4−/− mice exhibited a higher ΔT (P < 0.01) at 0700–0730 h and 1300–1330 h and a lower ΔT (P < 0.01) at 0100–0130 h than the control mice (Fig. 4f).

Tail suspension test

The time of immobility during a 6-min test period was assessed as a measure of depression-like behavior and expressed as percentage of the test duration. Analysis of variance showed that the three genotypes of mice investigated here differed significantly in this parameter (F2,48 = 19.87, P < 0.001). Specifically, the time that Y4−/− and Y2−/− mice spent immobile was significantly less than that spent immobile by control mice (Fig. 5).

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Figure 5. Behavior of control, Y4−/− and Y2−/− mice in the TST. The graph shows the time of immobility during a 6-min test period and is expressed as percentage of the test duration. The values represent means ± SEM, n as indicated in brackets. **P < 0.01 versus control mice.

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Circulating corticosterone levels

The baseline plasma levels of corticosterone determined in control, Y4−/− and Y2−/− mice that stayed in their home cage undisturbed until the time of trunk blood collection did not differ significantly between the three genotypes. Exposure to a moderate restraint stress at room temperature for 30 min caused a significant rise of circulating corticosterone in all mouse genotypes under study (anova for factor treatment: F1,27 = 89.87, P < 0.001). This stress-induced increase in the plasma levels of corticosterone was similar in control, Y4−/− and Y2−/− mice (Fig. 6).

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Figure 6. Corticosterone levels in blood plasma of control, Y4−/− and Y2−/− mice determined at baseline or 30 min after the end of a 30-min exposure to restraint stress. The values represent means ± SEM, n as indicated in brackets. **P < 0.01 versus respective levels at baseline. There were no genotype-related statistically significant differences.

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Object recognition test

Figure 7 presents the data of memory performance 6 h after initial object exposure. Analysis of variance showed that the performance in the ORT, expressed by the MI, differed significantly with the genotype (F2,21 = 6.299, P = 0.007). Post hoc analysis showed that the performance of Y2−/− mice in the object recognition task was significantly impaired relative to that of control animals, whereas the performance of Y4−/− mice was at least as good as that of control mice (Fig. 7). The relative time that the mice spent exploring one or the other object in the initial acquisition phase did not differ significantly in any of the genotypes investigated (data not shown).

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Figure 7. Behavior of control, Y4−/− and Y2−/− mice in the ORT. The graph shows the MI that was calculated according to the formula MI = (tn − to)/(tn + to), where to represents the time exploring the familiar object and tn represents the time exploring the novel object. The values represent means ± SEM, n as indicated in brackets. *P < 0.05 versus control mice.

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Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

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.

Neuropeptide Y as well as Y2 and Y4 receptors are present in the hypothalamus including the paraventricular nucleus (Dumont et al. 1998; Fetissov et al. 2004; Parker & Herzog 1999) in which knockout of the Y2 receptor causes a decrease in corticotropin-releasing factor (CRF) messenger RNA expression (Sainsbury et al. 2002b). There is evidence that NPY also interacts with CRF in the amygdala (Heilig 2004; Sajdyk et al. 2004, 2006) and that emotional–affective behavior is regulated by both extrahypothalamic and hypothalamic CRF, the latter controlling HPA axis activity (Cryan & Mombereau 2004; Holsboer 2000; de Kloet 2003; Shekhar et al. 2005). Because neither baseline nor stress-induced release of corticosterone, the neuroendocrine end-point of HPA axis activity, was altered by Y4 or Y2 receptor knockout, the behavioral alterations in Y4−/− and Y2−/− mice appear to be unrelated to alterations in HPA axis activity. This claim needs to be substantiated, however, by a more detailed analysis of the release profile of corticosterone (Müller et al. 2003; Oshima et al. 2003).

The similarity in the emotional traits of Y2−/− and Y4−/− mice raises the question as to the location of the Y4 and Y2 receptors involved and the nature of their endogenous ligand. While Y2 receptors have high affinity for NPY and peptide YY, Y4 receptors are particularly sensitive to pancreatic polypeptide (PP) (Lundell et al. 1996; Michel et al. 1998; Redrobe et al. 2004a). Neuropeptide Y has anxiolytic and antidepressant actions that are primarily mediated by Y1 receptors (Heilig 2004; Kask et al. 2002; Primeaux et al. 2005; Redrobe et al. 2002; Sajdyk et al. 2004, 2006). The anxiolytic- and antidepressant-like effect of Y2 receptor knockout is most probably because of deletion of presynaptic Y2 receptors, which will disinhibit the release of NPY and other transmitters and thus lead to an increased drive at Y1 receptors (Heilig 2004; Redrobe et al. 2003; Sajdyk et al. 2004; Tschenett et al. 2003). Microinjection experiments (Sajdyk et al. 2002) and region-specific deletion of Y2 receptors (Tasan et al. 2007) indicate that the action of Y2 receptors to modify anxiety- and depression-like behavior takes place in the amygdala.

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.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
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Acknowledgments

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

This study was supported by the Zukunftsfonds Steiermark (grant 262), the Austrian Scientific Research Funds (FWF grants L25-B05 and S-10204) and the Deutsche Forschungsgemeinschaft (grant KFO 125/1-1). The authors thank Professor Günther Sperk (Medical University of Innsbruck, Austria) for critically reading the manuscript, Dr Michael Trappitsch (Graz, Austria) for help in the development of the TST software and Dr Christoph K. Thoeringer (Max Planck Institute of Psychiatry, Munich, Germany) for advice on the behavioral experiments. The support by the director and staff of the Center for Medical Research (ZMF I) of the Medical University of Graz is greatly appreciated.