Elevated anxiety and antidepressant-like responses in serotonin 5-HT1A receptor mutant mice

Abstract

The brain serotonin (5-hydroxytryptamine; 5-HT) system is a powerful modulator of emotional processes and a target of medications used in the treatment of psychiatric disorders. To evaluate the contribution of serotonin 5-HT1A receptors to the regulation of these processes, we have used gene-targeting technology to generate 5-HT1A receptor-mutant mice. These animals lack functional 5-HT1A receptors as indicated by receptor autoradiography and by resistance to the hypothermic effects of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). Homozygous mutants display a consistent pattern of responses indicative of elevated anxiety levels in open-field, elevated-zero maze, and novel-object assays. Moreover, they exhibit antidepressant-like responses in a tail-suspension assay. These results indicate that the targeted disruption of the 5-HT1A receptor gene leads to heritable perturbations in the serotonergic regulation of emotional state. 5-HT1A receptor-null mutant mice have potential as a model for investigating mechanisms through which serotonergic systems modulate affective state and mediate the actions of psychiatric drugs.

Figure 1

(A) A schematic representation of the 5-HT_1A receptor gene, targeting construct, and the mutant allele. The region corresponding to the probe used for Southern blotting is indicated. (B) A representative Southern blot of BamHI-digested ES cell genomic DNA; the wild-type allele is indicated by a 12.3-kb band and the homologous recombinant (HR) allele indicated by a 6.5-kb band. (C) Southern blot of mouse tail genomic DNA from wild type (+/+), heterozygous (+/−), and homozygous mutant (−/−) animals. HSV-TK, herpes simplex virus thymidine kinase gene; BS, pBluescript SK vector; NEO, neomycin resistance cassette; B, BamHI; H, HindIII; S, SalI; P, PstI.

Figure 2

(A) Northern blot analysis of 5-HT_1A receptor gene expression in wild-type (+/+), heterozygous (+/−), and homozygous mutant (−/−) animals. A cyclophilin probe demonstrated equivalent RNA loading in all lanes. (B) [³H]8-OH-DPAT autoradiography of representative sections at the level of the septal nuclei (Top), mid-hippocampal formation (Middle), and dorsal raphe nucleus (Bottom). Anterior/posterior coordinates in mm relative to bregma are indicated, in accordance with the mouse-brain atlas of Franklin and Paxinos (39).

Figure 3

Immunohistochemical analysis of monoaminergic neurons in wild-type (+/+) and homozygous mutant (−/−) brains. Dorsal raphe serotonin neurons labeled with an anti-serotonin antibody (α-5-HT). Darkfield view of serotonergic innervation of the dorsal hippocampus labeled with an anti-serotonin transporter antibody (α-SERT). Dopaminergic neurons of the substantia nigra (SN) and ventral tegmental area (VTA) labeled with an anti-tyrosine hydroxylase antibody (α-TH). Noradrenergic neurons of the locus coeruleus (LC) labeled with α-TH. PY, CA1 pyramidal layer; SR, striata radiatum; LM, lacunosum moleculare; DM, dentate gyrus molecular layer; GR, dentate gyrus granule-cell layer.

Figure 4

Locomotor behavior in the open field. Mean (±SEM) time (A), distance (B), and entrances (C) into the central region of an open field during a 30-min exposure. Significant differences by genotype are indicated as ∗∗∗, P ≤ 0.001; ∗∗, P ≤ 0.01; ∗, P ≤ 0.05.

Figure 5

Behavior in the elevated-zero maze. Mean (±SEM) time (A), distance (B), open-quadrant entrances (C) and head dips (D) in the open quadrants of an elevated-zero maze during a 5-min trial. Significant differences by genotype are indicated as ∗∗∗, P ≤ 0.001; ∗∗, P ≤ 0.01; ∗, P ≤ 0.05.

Figure 6

Responses to a novel object. Mean (±SEM) (A) latency to approach a novel object placed in a familiar environment; (B) number of entries into area containing a novel object during a 30 min trial; (C) horizontal and (D) vertical activity during 30-min novel-object exposure. Significant differences by genotype are indicated as ∗∗∗, P ≤ 0.001; ∗∗, P ≤ 0.01; ∗, P ≤ 0.05.

Figure 7

Immobility in the tail-suspension assay. Mean (±SEM) time immobilized during the 6-min test. Significant differences by genotype are indicated as ∗∗∗, P ≤ 0.001; ∗, P ≤ 0.05.

Figure 8

Thermoregulatory responses to the administration of 8-OH-DPAT. Mean (±SEM) reduction in body temperature 20 min posttreatment (vehicle or 0.05, 0.2, 1.0 mg/kg 8-OH-DPAT; SC). Significant differences are indicated as ∗∗∗, P ≤ 0.001; ∗∗, P ≤ 0.01.