Attenuated response to stress and novelty and hypersensitivity to seizures in 5-HT4 receptor knock-out mice

Abstract

To study the functions of 5-HT4 receptors, a null mutation was engineered in the corresponding gene. 5-HT4 receptor knock-out mice displayed normal feeding and motor behaviors in baseline conditions but abnormal feeding and locomotor behavior in response to stress and novelty. Specifically, stress-induced hypophagia and novelty-induced exploratory activity were attenuated in the knock-out mice. In addition, pentylenetetrazol-induced convulsive responses were enhanced in the knock-out mice, suggesting an increase in neuronal network excitability. These results provide the first example of a genetic deficit that disrupts the ability of stress to reduce feeding and body weight and suggest that 5-HT4 receptors may be involved in stress-induced anorexia and seizure susceptibility.

Figure 1.

Targeted mutation of the 5-HT₄ receptor gene. a, Schematic diagram of a 6.5 kb 5-HT₄ genomic fragment encoding for the transmembrane domains II and III of 5-HT₄ receptors (exon III). The solid line (P) represents the external probe to detect Ase I-hybridizing restriction fragments (4 kb) from wild-type genomic DNA. b, Schematic representation of the 5-HT₄-targeting vector. The neomycin phosphotransferase gene (Neo) under the control of the phosphoglycerate kinase I promoter (pGK) is inserted in an engineered-created XbaI site localized in the DNA sequence encoding for the transmembrane domain III. The size of Ase I-hybridizing restriction fragments is 3.5 kb to identify the knock-out genomic DNA. c, Southern blot of genomic DNA from embryonic stem cells digested with Ase I and hybridized by the external probe.

Figure 2.

Radiograms of 5-HT₄ receptor-binding sites labeled with [³H]GR113808 in the frontal brain sections from wild-type, heterozygoous, and 5-HT₄ receptor-null mice. In wild-type animals, the [³H]GR113808 binding is more intense in the nucleus accumbens shell than the core, as analyzed previously in rat²⁴. At the same levels, the olfactory tubercles and fundus striati, like the striatum tail in a next posterior level, exhibit strong labeling. The various hypothalamic nuclei show moderate density (medial preoptic area). The concentration of binding sites covered an intermediate range, although a classic distinct laminar pattern is observed in the hippocampal formation: it is weak in the dorsal and medial raphe nuclei.

Figure 3.

The body weight of 5-HT₄ receptor-null and wild-type mice is similar during development. a, Data are mean ± SEM in body weight gain for a group of 20 wild-type and knock-out animals. Repeated measures ANOVA revealed no significant difference in body weight between mice of both genotypes from day 21 to 52 after birth. b-f, No significant difference between wild-type (n = 14) and 5-HT₄ receptor-null (n = 13) mice has been found at 4 months of age in body weight (b), feeding (c), water intake (d), or metabolism (e,f).

Figure 4.

In 5-HT₄ receptor-null mice, the restraint stress lost its ability to decrease food intake and body weight. Data are means ± SEM daily total food intake and body weight gain or loss for groups of 14 wild-type (WT) and 18 WT mice plus stress (a,c), 17 knock-out (KO), and 16 KO animals plus stress (b,d). Data were measured from the first day (9:00 A.M. in the light cycle) for the habituation (8 d) and recovery periods (10 d). The 110 min acute restraint stress or immobilization was applied on day 8 (arrow). Repeated measures ANOVA indicated significant effects of stress on food intake over the recovery period (F_(9,567) = 13.99; p < 0.0001) and an interaction of genotype and time (F_(9,549) = 2.2; p < 0.05). Subsequently, ANOVA analysis for each genotype and day of experiment revealed a significant difference in food intake between genotypes 24 hr after stress (F_(1,61) = 6.73; p < 0.05). a, Stress-induced anorexia in WT animals for the 48 hr recovery period. b, This effect is less marked in KO mice. In parallel, repeated measures ANOVA revealed significant effects of stress on body weight gain over the recovery period (F_(9,522) = 11.33; p < 0.0001) and an interaction of genotype and time (F_{(9, 522)} = 2.38; p < 0.05). c, d, Stress-induced, significant marked decreases in body weight gain in WT mice (c) but less or not effective in KO mice (d). Significant differences between restrained and unrestrained animals are marked (§§§p < 0.0001; §§p < 0.001; §p < 0.05). Significant genotype effect is noted (^*p < 0.05), and significant genotype x stress interaction is noted (#p < 0.05).

Figure 5.

The absence of 5-HT₄ receptors did not alter a stress-induced increase in corticosterone levels. Data are mean ± SEM corticosterone levels in wild-type (n = 9) and mutant mice (n = 12) 24 hr before and 30 min after a 5 min trial in the elevated plus maze.

Figure 6.

5-HT₄ receptor-null mice are less reactive in the open field than wild-type animals. a-e, Naive wild-type (open bars or points) and mutant (filled bars or points) animals were placed in identical open fields, and their horizontal activity (a-d) and time in the center (e) were monitored for 30 min (5 min per point) for three consecutive days. a, The mutant males were less active in the open field for the first 15 min of day 1 in the open field as compared with wild-type mice, whereas this effect was not detected over the following days. b, In the same line, the total distance traveled was lower in mutant animals than wild-type mice on the first day of exposure but not day 2 or 3. c, d, The absence of significant difference in the center/total path ratio between mice of both genotypes (d) indicated that this lower activity was detected in any part of the open field, as illustrated for the center (c). The mutant mice spent less time in the center during the first two consecutive days (e), which suggests a slight hyperanxiety-like behavior in the absence of 5-HT₄ receptors. Significant genotype effect is noted (^*p < 0.05). Significant differences in path between days for the wild-type or null mice are noted (§§p < 0.01; §p < 0.05).

Figure 7.

The 5-HT₄ receptor-null mice are hypersensitive to the convulsant GABA_A receptor PTZ compared with wild-type animals (open bars). a, b, Data are means ± SEM latency (s.) after the PTZ administration-induced tonic seizures (a) and death (b) in mice of both genotypes. Significant genotype effect is noted (^*p < 0.05).