Loss of the limbic mineralocorticoid receptor impairs behavioral plasticity

Abstract

Corticosteroid action in the brain is mediated by the mineralocorticoid (MR) and the glucocorticoid (GR) receptor. Disturbances in MR- and GR-mediated effects are thought to impair cognition, behavior, and endocrine control. To assess the function of the limbic MR in these processes, we inactivated the MR gene in the forebrain of the mouse using the Cre/loxP-recombination system. We screened the mice with a limbic MR deficiency in various learning and exploration tests. The mutant mice show impaired learning of the water-maze task and deficits in measures of working memory on the radial maze due to behavioral perseverance and stereotypy. They exhibit a hyperreactivity toward a novel object but normal anxiety-like behavior. The behavioral changes are associated with abnormalities of the mossy fiber projection and an up-regulation of GR expression in the hippocampus. Adult mutant mice show normal corticosterone levels at circadian trough and peak. This genetic model provides important information about the consequences of a permanently altered balance between limbic MR and GR, with implications for stress-related neuroendocrine and neuropsychiatric diseases.

Fig. 1.

Generation of mice lacking MR in the forebrain. (a) Organization of the mouse MR wild-type locus around exon 3. This exon was flanked with loxP sites by homologous recombination in embryonic stem cells. Transient expression of Cre recombinase in embryonic stem cells resulted in removal of the selection cassette (neomycin resistance–thymidine kinase), generating the MR^flox allele. This allele encodes an active MR protein but is recombined in any cell expressing Cre recombinase. Recombination results in deletion of exon 3 and thereby in inactivation of this allele (MR^null allele). The scheme shown depicts the wild-type locus, the targeting vector, and the resulting alleles. Triangles represent loxP sites, the filled box represents exon 3, open boxes represent the probes used for Southern blot analysis, and the arrows represent primers used for genotyping by PCR. E, EcoRV; S, SpeI. (b–p) MR protein expression in control animals (b, e, h, k, and n) and Cre expression (d, g, j, m, and p) and the corresponding loss of MR expression (c, f, i, l, and o) in MR^CaMKCre mice as revealed by immunohistochemistry on vibratome sections of 12-day-old animals. Depicted are the regions that show highest expression of MR in controls: lateral septum and indusium griseum (b–d), CA1 (e–g), CA2 and CA3 (h–j), dentate gyrus (k–m), and central amygdala (n–p).

Fig. 2.

Altered mossy fiber projections and hippocampal GR up-regulation in adult MR^CaMKCre mice. (a and b) Timm stain labeling of the Zn²⁺-containing mossy fiber terminals. Compared with control animals (a), MR^CaMKCre mice showed an aberrant layout of mossy fiber projections (b): variable excess of supragranular boutons in the dentate gyrus (single arrowheads), extension of the infrapyramidal mossy fiber field to the tip of CA3 and beyond into CA2 (double arrowheads), and a fuzzy distal boundary of the suprapyramidal field with boutons spreading across the pyramidal cell layer into CA2 and CA1 (asterisk). (c and d) Hippocampal GR expression as revealed by immunohistochemistry. GR is up-regulated in the cornu ammonis of adult MR^CaMKCre mice (d) compared with controls (c), most apparently in CA2 and CA3.

Fig. 3.

Normal basal circadian HPA-axis activity in MR^CaMKCre mice. (a and b) Plasma corticosterone levels of adult male (a) and female (b) MR^CaMKCre mice and their control littermates at diurnal trough (morning) and peak (evening), as well as after 40 min of restraint stress, showed no significant differences between both genotypes.

Fig. 4.

Impaired learning of MR^CaMKCre mice in a water-maze place navigation task with reversal and in a working memory procedure on the radial maze. (a) Place navigation: Training performance (six trials per day, blocks of two trials, cumulative search error in m·s). Despite reduced average performance, MR^CaMKCre mice (n = 33, controls = 31) showed significant learning at indistinguishable rates compared with controls during place acquisition as well as during place reversal. During subsequent cue navigation, the tested subset of MR^CaMKCre mice (n = 16, controls = 16) outperformed the control mice (which showed a transient wall-hugging response) during the first trial block, but the performances became indistinguishable during the second block. (b) Place navigation: Probe trial (% time in quadrant). Both groups spend significantly more time in the trained quadrant. (c) Place navigation: Probe trial. Both groups crossed the trained goal annulus significantly more often than control annuli in the adjacent quadrants. (d) Place navigation: Path plots of trials 19–30 (days 4 and 5, place reversal). A MR^CaMKCre mouse showing perseverative searching for the old goal site. Plots of a representative control mouse are shown for comparison. Filled square, actual goal; open square, previous goal; filled dot, begin; open dot, end of path. (e) Radial maze: Number of correct choices in the first eight. Both groups (n = 27, controls = 30) showed indistinguishable learning rates and performed above chance, but the average performance of MR^CaMKCre mice was clearly inferior. (f) Radial maze: Reentry errors. Whereas controls showed significant learning, no significant reduction of errors could be observed in MR^CaMKCre mice. Filled circles/columns, MR^CaMKCre; open circles/columns, control.

Fig. 5.

MR^CaMKCre mice (n = 31, controls = 33) were hyperreactive in an object exploration task but showed normal anxiety- and exploration-related behaviors in other tests. (a) Open-field. Zone preferences were not affected by the genotype. Chance levels for percent time depended on relative zone size and are shown by a dotted line. (b) Elevated O maze. Sector preferences were not affected by the genotype. Chance levels for percent time depended on relative zone size and are shown by a dotted line. (c) Light–dark box (% time in dark). Both groups preferred the dark box over the lit compartment to the same degree. (d) Emergence test (distance moved in m/min). The level of activity and the rate of habituation were not affected by the genotype. (e) Object exploration test. MR^CaMKCre mice displayed more than two times greater horizontal exploratory activity toward the object (m/min). (f) Object exploration test. MR^CaMKCre mice showed nearly three times as much estimated vertical exploratory activity toward the novel object (x/min) as did controls.