Evidence for the involvement of the kainate receptor subunit GluR6 (GRIK2) in mediating behavioral displays related to behavioral symptoms of mania

Abstract

The glutamate receptor 6 (GluR6 or GRIK2, one of the kainate receptors) gene resides in a genetic linkage region (6q21) associated with bipolar disorder (BPD), but its function in affective regulation is unknown. Compared with wild-type (WT) and GluR5 knockout (KO) mice, GluR6 KO mice were more active in multiple tests and super responsive to amphetamine. In a battery of specific tests, GluR6 KO mice also exhibited less anxious or more risk-taking type behavior and less despair-type manifestations, and they also had more aggressive displays. Chronic treatment with lithium, a classic antimanic mood stabilizer, reduced hyperactivity, aggressive displays and some risk-taking type behavior in GluR6 KO mice. Hippocampal and prefrontal cortical membrane levels of GluR5 and KA-2 receptors were decreased in GluR6 KO mice, and chronic lithium treatment did not affect these decreases. The membrane levels of other glutamatergic receptors were not significantly altered by GluR6 ablation or chronic lithium treatment. Together, these biochemical and behavioral results suggest a unique role for GluR6 in controlling abnormalities related to the behavioral symptoms of mania, such as hyperactivity or psychomotor agitation, aggressiveness, driven or increased goal-directed pursuits, risk taking and supersensitivity to psychostimulants. Whether GluR6 perturbation is involved in the mood elevation or thought disturbance of mania and the cyclicity of BPD are unknown. The molecular mechanism underlying the behavioral effects of lithium in GluR6 KO mice remains to be elucidated.

Figure 1

GluR6 KO mice have no significant impairment in the passive avoidance test. Acquisition (learning) and retention (memory) of the passive avoidance response did not differ in GluR6 KO and WT mice. During the training trial, all mice promptly entered the dark compartment, whereas after receiving foot shocks, all mice quickly learned to avoid entering the dark chamber (previously paired with foot shock), as indicated by increased latencies during the post-shock test trials. No statistical difference was found between the two groups in the avoidance latencies in the pre-shock trial and during retention trials conducted 5 min, 1 day, 2 days and 15 days post-shock (two-way ANOVA: genotype effect, F_1,50 = 2.75, P > 0.1 (NS)). Results are means±s.e.m.

Figure 2

GluR6 KO mice exhibit increased spontaneous activity in the open field and home cage. (a) To study spontaneous locomotion in the open field, mice were placed in one corner of an open-field arena and their behavior was recorded for 60 min. GluR6 KO mice displayed increased spontaneous locomotor activity during 1 h of the first day of the open-field test compared with GluR5 KO and WT mice (two-way ANOVA, genotype effect, F_2,35 = 87.55, P < 0.0001). (b) GluR6 KO mice displayed persistent hyperactivity in the open-field arena at each trial conducted over 3 consecutive days and also at days 14 and 15 from the first exposure (two-way ANOVA: genotype F_2,174 = 148.3, P < 0.0001; day F_4,174 = 4.9, P < 0.001; genotype × day F_8,174 = 1.2, P = NS and one-way ANOVA (day): GluR6 KO F_4,60 = 1.0, P = NS; GluR5 KO F_4,60 = 4.6, P < 0.01; WT F_4,54 = 3.6, P = 0.01). To study the home cage activity, WT and GluR6 KO mice were monitored in their home cages using a digital camcorder for 1 h between 0800 and 1000 hours for 2 consecutive days. Digital video files were analyzed using CleverSystems' HomeCageScan Suite for general activity (including, turning, walking, digging, grooming, drinking and eating) and exploratory activity. (c) GluR6 KO mice displayed significantly more home cage general activity, which was consistent over 2 days of testing (two-way ANOVA: genotype F_1,14 = 8.194, P = 0.0125; time F_1,14 = 1.926, P = 0.1869; genotype × time F_1,14 = 0.02712, P = 0.8715). (d) GluR6 KO mice also displayed significantly more home cage exploratory activity, which was consistent over 2 days of testing (two-way ANOVA: genotype F_1,14 = 10.99, P = 0.0051; time F_1,14 = 1.279, P = 0.2771; genotype × time F_1,14 = 0.3059, P = 0.5890). Results are means±s.e.m. *P ≤ 0.05.

Figure 3

GluR6 KO mice exhibit increased amphetamine-induced response. (a) To study injection-induced irritability, mice were first placed in one corner of the arena and their behavior was recorded for 30 min as a control for amphetamine injection; immediately afterwards they were intraperitoneally injected with saline and placed back in the same arena for an additional 30 min. Saline injection did not affect locomotor activity for any of the three groups. (b) To study amphetamine-induced hyperactivity, the same procedure as in panel (a) was carried out, except that instead of saline, 2 mg kg⁻¹ amphetamine was administered. Amphetamine increased locomotor activity above 25–30 min activity levels in mice of all three genotypes. (c) GluR6 KO mice displayed significantly more increases above the 25–30-min activity level after amphetamine injection compared with WT and GluR5 KO mice (ANOVA for genotype, F_2,32 = 5.529, P = 0.0087; Tukey's multiple comparison test, WT vs GluR6 KO mice, P < 0.05; GluR5 KO vs GluR6 KO mice, P < 0.05). Results are means±s.e.m. *P≤0.05.

Figure 4

GluR6 KO mice display robustly aggressive behavior. (a) In the social interaction test, pairs of mice within the same strain were placed in opposite corners of an open-field arena. The frequency of social behaviors was counted over 5 min. GluR6 KO mice displayed a high frequency of destructive (aggressive) behaviors compared with GluR5 KO and WT mice (one-way ANOVA for ‘tail rattling’: F_2,19 = 11.4, P < 0.001; ‘attack bite’: F_2,19 = 3.9, P < 0.04). GluR6 KO mice displayed a high frequency of exploratory behavior compared with WT mice (one-way ANOVA for ‘sniffing’: F_2,19 = 3.44, P = 0.05; post hoc Fisher LSD test for ‘sniffing’: GluR6 KO vs WT mice, P < 0.03). GluR6 KO mice displayed the same side-by-side behavior as GluR5 KO and WT mice (one-way ANOVA for ‘side by side’ F_2,19 = 0.02, P = NS). (b) In the resident–intruder test, a 129Sv/Ev naive male mouse was introduced to the home cage of each mouse tested. The frequency of attacks on the intruder was counted over 5 min. GluR6 KO mice exhibited a high frequency of attack events toward their intruder compared with WT mice, who exhibited no attacks toward their intruder (Student's t-test: t₂₃ =−2.12, P < 0.05). Results are means±s.e.m. *P≤0.05.

Figure 5

GluR6 KO mice show increased activity in anxiety-provoking subregions of the open-field test. To further study the activity in an anxiety-provoking environment, mice were placed in one corner of an open-field arena; the frequency of entering events to the center of the arena and the total time spent in it was recorded over 60 min. (a) GluR6 KO mice displayed a higher frequency of entries to the center of the open-field arena compared with GluR5 KO or WT mice (one-way ANOVA: F_2,35 = 33.6, P < 0.0001). (b) GluR6 KO mice increased their time spent in the center of the open-field arena compared with GluR5 KO or WT mice (one-way ANOVA: F_2,35 = 13.4, P < 0.0001). Results are means±s.e.m. *P < 0.0001.

Figure 6

GluR6 KO mice exhibited increased activity in anxiety-provoking regions of the elevated plus maze test. A plus-shaped maze elevated above ground containing two dark closed arms and two open and lit arms without walls was used to examine activity in anxiety-provoking regions. Each mouse was placed in the center of the maze; time and frequency of visits to the different zones of the maze, as well as locomotion measures, were collected for 5-min sessions. (a) Ratios of open-arm vs total (open and closed arms) duration of time spent. GluR6 KO mice exhibited increases in duration of time spent in the open arms of the maze (ANOVA: F_2,16 = 5.783, P = 0.0129; WT vs GluR6 KO mice, P < 0.05; WT vs GluR6 KO mice, P < 0.05). (b) Ratios of open-arm vs total arm entries. GluR6 KO mice exhibited increased open-arm entries (ANOVA: F_2,16 = 6.256, P = 0.0098; WT vs GluR6 KO mice, P < 0.05; WT vs GluR6 KO mice, P < 0.05). (c) Ratios of open-arm vs total arm distance traveled. GluR6 KO mice exhibited increased distance traveled in the open arms (ANOVA: F_2,16 = 7.264, P = 0.0057; WT vs GluR6 KO mice, P < 0.05; WT vs GluR6 KO mice, P < 0.05). Results are means±s.e.m. *P < 0.05.

Figure 7

GluR6 KO mice displayed decreased immobility time in the forced swim test. Mice were placed in cylinders filled with water in a way that they were not able to touch the floor or escape for 6 min. Immobility time, defined as a lack of activity aside from small movements needed to keep the body floating, was measured throughout the last 4 min of the session. Immobility time was reduced for GluR6 KO mice compared with GluR5 KO or WT mice (one-way ANOVA: F_2,35 = 4.5762, P < 0.05). Results are means±s.e.m. *P < 0.05.

Figure 8

Chronic lithium treatment reduced spontaneous locomotor activity in GluR6 KO mice. (a) Four weeks of lithium treatment attenuated the spontaneous locomotor activity of GluR6 KO mice studied over 3 consecutive days of open-field test (two-way ANOVA: genotype F_3,57 = 647.7, P < 0.0001; day F_2,57 = 1512, P = NS; genotype×day F_6,57 = 0.8, P = NS; one-way ANOVA: day 1: F_3,19 = 7.7, P < 0.01; day 2: F_3,19 = 25.7, P < 0.001; day 3: F_3,19 = 14.2, P < 0.001; post hoc Fisher LSD test: P < 0.05, P < 0.001). Results are mean percentage of WT total activity of the same day. (b) Effects of 4-day lithium treatment on spontaneous locomotor activity of GluR6 KO mice. Short-term lithium treatment did not significantly alter spontaneous locomotor activity of GluR6 KO mice. Data are percent of mean value of GluR6 KO mice treated with control food. Results are means±s.e.m. *P≤0.05.

Figure 9

Chronic lithium dramatically decreased aggressive behavior in GluR6 KO mice. (a) Social interaction test: 4 weeks of lithium treatment caused a complete loss of tail rattling and attack bite events displayed by GluR6 KO mice toward their opponents from the same group (one-way ANOVA: tail rattling: F_3,8 = 6.9, P < 0.05; attack bite F_3,8 = 3.9, P = 0.05; sniffing: F_3,8 = 1.3, P = 0.3 (NS); side by side: F_3,8 = 0.63, P = 0.6 (NS)). (b) Resident–intruder test: 4 weeks of lithium treatment caused a robust reduction in attacks displayed by GluR6 KO mice toward a naive male intruder of the same strain (one-way ANOVA: F_3,19 = 3.2, P < 0.05). Results are mean of percentage relative to WT±s.e.m. Li, lithium (chronic treatment). (c) Effects of 6 days of lithium treatment on measures of the resident–intruder test in GluR6 KO mice. Short-term lithium treatment did not significantly reduce attack frequencies of GluR6 KO mice (t₁₄ = 1.171, P = 0.2611). Results are means±s.e.m. *P < 0.05.

Figure 10

Chronic lithium treatment attenuated increased activity in anxiety-provoking regions. (a) Frequency of entries to the center of the arena during the first day of the open-field test. Four weeks of lithium treatment reduced the frequency of entries to the center of the open-field arena in GluR6 KO mice (one-way ANOVA: F_3,19 = 9.8, P < 0.001; post hoc Fisher LSD test: GluR6 KO vs GluR6 KO+Li, WT+Li or WT mice, P < 0.05; GluR6 KO+Li vs WT+Li or WT mice, P = 0.01). (b) Duration of time spent in the center of the arena during the first day of open-field test. Four weeks of lithium treatment reduced the amount of time spent in the center of the open-field arena in GluR6 KO mice (one-way ANOVA: F_3,19 = 9.2, P < 0.001; post hoc Fisher LSD test: GluR6 KO vs GluR6 KO+Li, WT+Li or WT mice, P < 0.05; GluR6KO+Li vs WT mice, P = 0.01). Results are means of percentage relative to WT±s.e.m. Li, lithium (chronic treatment). The effects of 5 days of lithium treatment on the increased activity in anxiety-provoking regions of the open-field test in GLuR6 KO mice were also studied. (c) The frequency of center entries of GluR6 KO mice was not reduced by short-term lithium treatment. (d) The time that GluR6 KO mice spent in the center of the arena was also not reduced by short-term lithium treatment. Results are means±s.e.m. *P≤0.05.

Figure 11

Effects of chronic and short-term lithium treatment on measures of the elevated plus maze test. Chronic (4 weeks, (a–c)) and short-term (4 days, (d–f)) lithium treatment did not significantly alter duration of time spent in the open arms of the maze (a and d), frequency of open-arm entries (b and e) and distance traveled in the open arms (c and f) in GluR6 KO mice. Results are means±s.e.m.

Figure 12

Chronic lithium treatment further reduced GluR6 KO and WT mice immobility time in the forced swim test. Both GluR6 KO and WT mice reduced their immobility time in the forced swim test by more than 60% after chronic lithium treatment (two-way ANOVA: genotype F_1,19 = 6.6, P < 0.02; treatment F_1,19 = 13.5, P < 0.01 with no interaction). Results are means of percentage relative to WT±s.e.m. Li, lithium (chronic treatment). *P < 0.01.

Figure 13

The GluR5 subunit membrane expression levels were reduced in the hippocampus and FCX of GluR6 KO mice. (a) The GluR5 protein levels were reduced in the hippocampal membrane fraction of the GluR6 KO mice (one-way ANOVA: F_2,22 = 51.1, P < 0.001; post hoc Fisher LSD test: GluR6 KO and GluR6 KO+Li vs WT mice, P < 0.001). Four weeks of chronic lithium treatment did not affect GluR5 protein level reduction in the hippocampal membrane fraction of the GluR6 KO mice (post hoc Fisher LSD test: GluR6 KO vs GluR6 KO+Li mice, P = 0.80 (NS)). (b) The GluR5 protein levels were reduced in the FCX membrane fraction of the GluR6 KO mice (one-way ANOVA: F_2,22 =18.159, P < 0.001; post hoc Fisher LSD test: GluR6 KO and GluR6KO+Li vs WT mice, P < 0.001). Four weeks of chronic lithium treatment did not affect GluR5 protein level reduction in the FCX membrane fraction of the GluR6 KO mice (post hoc Fisher LSD test: GluR6 KO vs GluR6 KO+Li mice, P = 0.92 (NS)). (c) The GluR5 protein levels were not reduced in the hippocampal cytosol fraction of GluR6 KO mice. Four weeks of chronic lithium treatment did not affect GluR5 levels in the hippocampal cytosolic fraction of GluR6 KO mice (one-way ANOVA: F_2,21 = 0.61, P = 0.55). (d) The GluR5 protein levels were not reduced in the FCX cytosol fraction of GluR6 KO mice. Four weeks of chronic lithium treatment did not affect the GluR5 levels in the FCX cytosolic fraction of GluR6 KO mice (one-way ANOVA: F_2,18 = 2.63, P = 0.10 (NS)). FCX, frontal cortex; Li, lithium (chronic treatment). Results are mean±s.e.m. in arbitrary units (AU). *P < 0.001.

Figure 14

KA-2 subunit membrane expression levels were reduced in the hippocampus and FCX of GluR6 KO mice. (a) The KA-2 protein levels were reduced in the hippocampal membrane fraction of GluR6 KO mice (one-way ANOVA: F_2,22 = 168.34, P < 0.001; post hoc Fisher LSD test: GluR6 KO and GluR6 KO+Li vs WT mice, P < 0.001). Four weeks of chronic lithium treatment did not affect KA-2 protein level reduction in the hippocampal membrane fraction of GluR6 KO mice (post hoc Fisher LSD test: GluR6 KO vs GluR6 KO+Li mice, P = 0.98 (NS)). (b) The KA-2 protein levels were reduced in the FCX membrane fraction of GluR6 KO mice (one-way ANOVA: F_2,22 = 84.14, P < 0.001; post hoc Fisher LSD test: GluR6 KO and GluR6 KO+Li vs WT mice, P < 0.001). Four weeks of chronic lithium treatment did not affect the KA-2 protein level reduction in the FCX membrane fraction of GluR6 KO mice (post hoc Fisher LSD test: GluR6 KO vs GluR6 KO+Li mice, P = 0.89 (NS)). (c) A trend toward increased KA-2 protein levels in the hippocampal cytosol fraction of GluR6 KO mice was observed; this trend was not affected by lithium treatment (one-way ANOVA: F_2,21 = 3.38, P = 0.053 (NS)). (d) A trend toward increased KA-2 protein levels in the FCX cytosol fraction of GluR6 KO mice was observed; this trend was not affected by lithium treatment (one-way ANOVA: F_2,18 = 1.37, P = 0.28 (NS)). FCX, frontal cortex; Li, lithium (chronic treatment). Results are mean±s.e.m. in arbitrary units (AU). *P < 0.001.