Mineralocorticoid and glucocorticoid receptor balance in control of HPA axis and behaviour

Psychoneuroendocrinology (2013) 38, 648—658

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Mineralocorticoid and glucocorticoid receptor balance in control of HPA axis and behaviour A.P. Harris a,*, M.C. Holmes a, E.R. de Kloet b, K.E. Chapman a, J.R. Seckl a a

Endocrinology Unit, Centre for Cardiovascular Science, The Queen’s Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK b Leiden Univ, Div Med Pharmacol, Leiden Amsterdam Ctr Drug Res LACDR, POB 9502, NL-2300 RA Leiden, Netherlands Received 11 June 2012; received in revised form 6 August 2012; accepted 15 August 2012

KEYWORDS Glucocorticoid receptor; Mineralocorticoid receptor; HPA axis; Cognition; Transgenic mice; Psychopathology

Summary An imbalance between central glucocorticoid (GR) and mineralocorticoid (MR) receptors is proposed to underlie the HPA axis dysregulation that associates with susceptibility to psychopathology (anxiety, PTSD). To test this ‘balance hypothesis’ we examined whether the impact of MR levels upon HPA-axis control and behaviour depended on the relative levels of GR and vice versa. Avoiding antenatal maternal ‘programming’ effects by using littermates, we generated mice with forebrain MR over-expression (MRhi) and/or simultaneous global GR underexpression (GRlo). We found a significant interaction between MR and GR in control of the HPA-axis under stressed but not basal conditions. With reduced GR levels, HPA-axis activity in response to restraint stress was enhanced, likely due to impaired negative feedback. However, high MR in concert with reduced GR minimised this HPA-axis overshoot in response to stress. MR:GR balance also played a role in determining strategies of spatial memory during a watermaze probe trial: when coupled with GR under-expression, MRhi show enhanced perseveration, suggesting enhanced spatial recall or reduced exploratory flexibility. Other alterations in cognitive functions were specific to a single receptor without interaction, with both MRhi and GRlo manipulations independently impairing reversal learning in spatial and fear memory tasks. Thus, MR and GR interact in specific domains of neuroendocrine and cognitive control, but for other limbicassociated behaviours each receptor mediates its own repertoire of responses. Since modulation of HPA-axis and behavioural dysfunction associated with high levels of MR, selective ligands or transcriptional regulators may afford novel therapeutic approaches to affective psychopathologies. # 2012 Elsevier Ltd. All rights reserved.

1. Introduction * Corresponding author. Tel.: +44 0131 242 6777; fax: +44 0131 242 6779. E-mail address: [email protected] (A.P. Harris).

An imbalance between central corticosteroid receptors is proposed to underlie dysregulation of the hypothalamic— pituitary—adrenal (HPA) axis and vulnerability to

0306-4530/$ — see front matter # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.psyneuen.2012.08.007

MR:GR balance and behaviour stress-related psychiatric disorders (e.g. anxiety, depression, post traumatic stress disorder [PTSD]) (e.g. De Kloet, 1991; Sapolsky, 2000). In the brain, glucocorticoids act through two receptors: the high affinity mineralocorticoid receptor (MR) and the lower affinity glucocorticoid receptor (GR). Both belong to the same family of intracellular ligand-dependent transcription factors. Though highly homologous, particularly in the DNA binding domain, there is limited overlap in the sets of target genes they regulate (Datson et al., 2001). The receptors are also found in the cell membrane where they exert fast, non-genomic effects on neuronal excitability (Joels et al., 2009; Karst et al., 2005). MR and GR are abundant in the limbic system (e.g. hippocampus, amygdala), circuitry essential for emotion, cognition and HPA axis control (Ahima and Harlan, 1990). MR and GR mediate the initiation (possibly via membrane MR; Karst et al., 2005) and termination of the HPA axis stress response (primarily via negative feedback at intracellular GR, but also MR) and modulate acquisition, processing, storage and retrieval of stressful experiences (e.g. Oitzl et al., 2001; Sapolsky et al., 2000). Imbalance between MR and GR-mediated actions in limbic system neurons may lead to an exaggerated or inadequate initial HPA axis response to stress, impaired containment, delayed recovery and compromised adaptation. Crucially, such changes are hypothesised to increase vulnerability to affective disease (De Kloet et al., 1998). Polymorphisms in genes encoding MR and GR associate with altered HPA axis function and depression, suggesting a causal role (van West et al., 2006; Wust et al., 2008). In mice, global reduction in GR signalling activates the HPA axis and impairs cognition (Oitzl et al., 2001; Ridder et al., 2005), while forebrain-selective deletion of GR increases corticosterone levels, reduces anxiety and increases depressive-like behaviour (Boyle et al., 2005; Tronche et al., 1999). In contrast, forebrain-specific deletion of MR impairs learning and memory (Berger et al., 2006), whereas forebrain overexpression of MR enhances memory and reduces anxiety (Lai et al., 2007; Rozeboom et al., 2007). Experiments that change the level of only one receptor fail to reveal if the observed effects are a consequence of the absolute level of the altered receptor, or the resulting imbalance between MR and GR. Here, to address the ‘balance’ hypothesis directly, we crossed mice underexpressing GR globally (modelling human GR haploinsufficiency) with mice over-expressing MR in the forebrain (avoiding changes in kidney that can alter salt balance and blood pressure). Importantly, these experiments were all carried out on mouse littermates (with each genetically identical dam bearing all genotypes), removing the confounding effects of developmental programming by the in utero and early postnatal environment which otherwise potently impact on HPA axis and affective functions (e.g. Harris and Seckl, 2011).

2. Materials and methods 2.1. Generation of mice Mice were generated by crossing male mice heterozygous for a null allele of GR, which have half the normal density of GR in brain (GRbgeo/+, here termed GRlo mice) (Michailidou et al.,

649 2008), with female mice over-expressing human MR in the forebrain under the control of the CamKIIa promoter (MRtg/+, here termed MRhi mice) (Lai et al., 2007), ensuring identical maternal environment for all experimental mice. Both parent strains were back-crossed to the C57BL/6J background >16 generations and maintained as hemizygotes. This cross generated a 1:1:1:1 ratio of the following genotypes: GR+/  MRtg/+ (GRloMRhi), GR+/MR+/+ (GRloMRnorm, where ‘norm’ indicates wild-type or ‘unaltered’ levels of receptor), GR+/ + MRtg/+ (GRnormMRhi), and GR+/+MR+/+ (GRnormMRnorm, i.e. wild type mice).

2.2. Genotyping and animal husbandry Genotyping of mice was carried out as described previously (Lai et al., 2007; Michailidou et al., 2008). All experiments were carried out on groups (N = 8—10 unless otherwise stated) of male mice housed two or three per cage (genotypes mixed within a cage) under controlled lighting (lights on 07:00—19:00 h) and fed standard chow diet ad libitum from weaning. Animal care and experimental protocols were conducted in strict accordance with Home Office and institutional guidelines.

2.3. HPA axis function Blood samples for measurement of corticosterone levels were collected by tail nick between 08:00 and 09:00 h (nadir levels), and at least one week later in the same animals between 20:00 and 21:00 h (peak levels). All mice were aged 7 m (months) and had previously undergone affective behavioural testing at 5—6 m. Measurement of plasma corticosterone levels following acute stress was performed in the same mice, approximately 3 weeks later. A blood sample was taken from a basal tail nick under non-stressed conditions between 09:00 and 12:00 h. Repeat blood samples were then taken following 40 min restraint in a clear Perspex tube (3 cm diameter with adjustable length) and again 1 h 40 min following onset of the restraint. Plasma corticosterone levels were measured using an in-house radioimmunoassay, as described elsewhere (Lai et al., 2007).

2.4. Behavioural testing All behavioural tests were conducted between 09:00 and 16.00 h. The observer was blind to genotype. 2.4.1. Spatial cognition: Y-maze Details and procedure for the Y-maze have been described elsewhere (Yau et al., 2007). Briefly, each mouse explored the maze for 5 min with one arm closed (novel arm) then returned to its home-cage. After an inter-trial interval (ITI) of variable length, each mouse was returned to the maze for a retrieval trial (2 min long) during which all arms were open for investigation. The time spent in the novel arm was calculated as percentage of total time in all three arms. A 1 min ITI was first used to confirm motivation to explore the novel arm and test vision. Seven days later mice were retested with a 2 h ITI (with different spatial cues) to measure spatial memory.

650 2.4.2. Spatial cognition: water maze (WM) Details of the water maze have been described elsewhere (Yau et al., 2001). All swim trials were recorded using LimeLightTM video tracking system and analysed using Actimetrics software (Actimetrics Inc., IL, USA). Mice were 6 m of age and had undergone affective behavioural testing approximately 10 days earlier. 2.4.3. Reference memory water maze protocol Mice were trained over 8 days to find a hidden escape platform in a fixed location with 4 swims per day (1 h inter-trial interval, ITI). Each mouse was released from one of four release points in a pre-determined random order. For each trial, mice had up to 90 s to find and mount the platform and were guided there if they failed to find it. Mice were left for 20 s before being removed from the platform, towel dried and placed back in the home cage. Latency, swim speed and distance were recorded as measures of learning. On day 9 (24 h after the last test), each mouse received a 60 s probe trial in which the platform was absent. The latency to reach where the platform had been during training, platform crossings (if the platform had been present) and the % dwell time in each of the four quadrants was measured. After 60 s the platform automatically rose from the bottom of the maze to the surface of the pool, thus preventing extinction of this platform location and permitting subsequent investigation of reversal learning in these mice. 2.4.4. Reversal learning protocol Following the above reference memory water maze protocol, each mouse received 4 swims per day (1 h ITI) for 4 days with the hidden platform located in the diagonally opposite quadrant to where the mouse had been previously trained. Latency to reach the platform and % dwell time in all quadrants was recorded. A long latency coupled with a high % dwell time in the old goal site during this training was considered a marker of impaired extinction learning. 2.4.5. Spatial versus visible platform protocol To investigate rigidity of strategy in the water maze, mice were given a trial in which the only escape was a visible platform (1 cm above water with Lego blocks on top) positioned in the diagonally opposite quadrant to where the mice had been spatially trained for the previous 8 days. The latency to reach the visible platform (maximum 90 s) and dwell times in each of the quadrants was recorded. A long latency to find the visible platform was considered a marker of inflexibility. To control for motivation and visual ability, in the absence of spatial cues, mice were given a trial in which the platform was visible the following day. A water maze in a different room was used to ensure removal of all familiar cues and curtains were drawn around the maze to remove extra-maze cues. The latency to reach the visible platform was recorded. 2.4.6. Fear memory formation and extinction: passive avoidance apparatus An inhibitory avoidance shuttle box (Ugo Basile, Milan, Italy) comprising two equal compartments (18 cm  10 cm  16 cm), each with a grid floor was used. One compartment was made from white opaque plastic and was illuminated (350 lx). The

A.P. Harris et al. other compartment was made from black plastic and not illuminated (4 lx). An automatic door separated the compartments. 2.4.7. Passive avoidance protocol A naı¨ve cohort of mice were subjected to the passive avoidance test. To ensure acclimatisation with the apparatus and procedure, mice were given a ‘no shock’ trial on day 1. Mice were individually placed in the light compartment (tail towards the door). After a 40 s delay, the door between the light and dark compartment opened automatically and the latency to cross completely into the dark compartment was recorded. Training to the conditioned passive avoidance reflex began approximately 3 h later when each mouse received its first training session in which transfer to the dark compartment was accompanied by a 0.3 mA foot shock for 3 s. 24 h later in the next trial the door opened after a delay of 5 s and upon transfer to the dark, mice received a 0.6 mA foot shock for 3 s. Extinction of the conditioned behaviour was induced by exposing the mice to the dark compartment without foot shock after transfer every day for 13 days. In all trials, the maximum period allowed for transfer to the dark was 300 s, after which time the mouse was gently guided into the dark compartment. After 10 s, the mice were removed from the dark compartment after each trial. To confirm that reduced latency upon repeated testing represents the active process of extinction (rather than forgetting), naı¨ve wild type mice were shocked as above and memory was tested on days 7 and 11 only, without any prior extinction trials. 2.4.8. Affective behavioural and nociception testing Mice were acclimatised to the experimental room for at least 24 h prior to testing. All affective behaviour trials were recorded using LimeLightTM video tracking system and analysed by Actimetrics software (Actimetrics Inc., IL, USA) (N = 10—16 per genotype). Naı¨ve mice were tested as follows: first, the elevated plus maze, second (at least 10 days later) the light/dark box and third (7 days later), the open field. Naı¨ve mice were subject to either the tail suspension test or the nociception test. 2.4.9. Elevated plus maze Mice were placed in the maze and allowed to explore for 5 min; decreased exploration of the open arms was indicative of anxiety-like behaviour. 2.4.10. Light/dark box Mice were placed in the dark compartment and given 5 min to explore; high latency to enter the light compartment (120 lx), was considered a marker of anxiety-like behaviour. 2.4.11. Open field Mice were placed in the centre of brightly lit arena (90 lx, 50 cm  50 cm  25 cm high) and allowed to explore for 5 min; decreased exploration of the centre was indicative of anxiety-like behaviour. 2.4.12. Tail suspension test To measure depressive-like behaviour, mice were suspended by the tail for 5 min (tail taped with surgical tape, 1 cm from

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the tip of the tail) 40 cm from the floor. Duration of immobility (completely passive hanging) was scored. 2.4.13. Nociception testing To establish that differences observed in avoidance learning and extinction were not due to altered pain threshold, nociception was tested using a hot plate. The hot plate was heated to 55 8C and each mouse was individually placed on the plate; a clear Perspex cylinder prevented escape. Latency for each mouse to lick a paw was noted (N = 5—7/ genotype).

2.5. Measurement of brain MR, GR and GILZ mRNA levels Naı¨ve mice were culled at approx 5 m of age and brains collected between 12:00 and 15:00 h. Coronal sections (10 mm) of frozen brains were used for in situ hybridisation using 35S-UTP (PerkinElmer) labelled RNA probes complimentary to mRNA encoding human MR or rat GR as described previously (Lai et al., 2007; Miesfeld et al., 1986). For each mouse, 3 sections of the dorsal hippocampus (CA1, CA3 and dentate gyrus [DG]) and the basolateral amygdala (BLA) were measured, and densitometric analyses performed as described elsewhere (Lai et al., 2007). Levels of mRNA encoding glucocorticoid induced leucine zipper (GILZ) were quantified by real-time PCR. Hippocampal RNA was extracted as previously described (Ridder et al., 2005) and real-time PCR was carried out on cDNA using a LightCycler 480 (Roche Diagnostics, Burgess Hill, UK) with a commercial master mix (FAM-hydrolysis probe, Roche Diagnostics) using forward 50 -TCCGTTAAACTGGATAACAG-30 and reverse primers 50 -TGGTTCTTCACGAGGTCCAT-30 primer with Universal Probe Library probe 49 (Roche). Levels of GILZ were normalised against hypoxanthine-phosphoribosyl transferase (HPRT) mRNA levels using forward 50 TCCTCCTCAGACCGCTTT-30 and reverse 50 -CCTGGTTCATCATCGCTAATC-30 primers with Universal Probe Library probe 95.

2.6. Statistical analysis Data are reported as mean  SEM. Repeated measures were analysed by repeated measures 2-way ANOVA using the statistical software package JMP. Between subject factors were ‘MR’ (with two levels: hi/norm), and ‘GR’ (with two levels: lo/norm), the with-in subject factor for in situ data was region (CA1, CA3, dentate gyrus and basolateral amygdala) and for cognitive and HPA axis testing was ‘time’. To correct for violations of sphericity (the assumption that the variance of the differences between repeated measures is equal over time), the Greenhouse—Geisser correction term was applied to the degrees of freedom and associated P values (thus, degrees of freedom are not always whole numbers). All other data were analysed using 2-way ANOVA in which a significant MR-by-GR interaction term indicates that the effect of one receptor depends on the level of the other, supporting the hypothesis that MR:GR balance is key. Significant main effects and interactions were analysed further with Bonferroni post hoc tests. All data were checked and found to be normally distributed and with no heterogeneity of variance.

3. Results 3.1. Alteration of MR or GR does not affect expression of the other MRhi mice had 3.2-fold higher level of MR mRNA expression (F 1,14 = 251.8, P < 0.0001) and GRlo mice had 50% reduced GR mRNA levels in the hippocampus (F 1,14 = 33.4, P < 0.0001; Table 1). MRhi mice also had an 8.5-fold increase in MR mRNA expression levels in the basolateral amygdala (BLA) (F 1,16 = 355.1, P < 0.0001; Table 1) and GRlo mice had 37% reduced GR mRNA levels in the BLA (F 1,15 = 9.2, P = 0.0085). Crucially, under-expression of GR (either alone or in concert with MRhi) did not alter co-localised expression of MR mRNA and, similarly, over-expression of MR (alone or in concert with GRlo) did not significantly affect co-localised GR mRNA levels in the hippocampus or amygdala.

Table 1 MR and GR mRNA levels measured by in situ hybridisation. N = 4—5/genotype and values are mean  SEM and GILZ mRNA levels in total hippocampus measured by real-time PCR. GRnormMR norm

GRnormMR hi

GRloMR norm

GRloMR hi

MR mRNA CA1 CA3 DG BLA

100.0  14.5 100.0  12.7 100.0  12.4 100.0  15.4

315.0  11.3 *** 278.4  13.5 *** 387.5  11.8 *** 809.2  52.5 ***

136.8  13.0 124.1  24.1 124.1  20.9 126.6  16.3

297.4  12.9 *** 277.8  8.8 *** 360.7  16.4 *** 890.0  47.4 ***

GR mRNA CA1 CA3 DG BLA

100.0  4.5 100.0  12.1 100.0  9.8 100.0  26.5

96.2  9.6 87.5  6.5 96.0  11.1 93.7  20.3

51.2  5.5 *** 40.7  8.3 *** 51.1  4.4 *** 44.4  15.1 ***

61.1  10.8 *** 51.6  8.0 *** 66.8  2.4 *** 29.4  11.8 ***

GILZ mRNA Total hippocampus

100.0  16.5

78.8  7

93.2  16.8

93.2  16.8

Mean  SEM values for mRNA expressed as % of wild type levels (BLA: basolateral amygdala, DG: dentate gyrus, CA: cornu ammonis). *** Significantly different to wild type (GRnormMRnorm).

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3.2. HPA axis activity in response to restraint stress Basal plasma corticosterone levels (morning) were unaffected by MR overexpression (F 1,22 < 0.01, P = 0.97) or GR underexpression (F 1,22 < 0.001, P = 0.89) nor was there a significant MR-by-GR interaction (Fig. 1A). Diurnal peak (evening) plasma corticosterone levels were significantly higher in GRlo mice (Fig. 1B; F 1,22 = 4.4, P = 0.048) as previously reported (Michailidou et al., 2008). MRhi alone or with GRlo had no impact on diurnal peak corticosterone levels (Fig. 1B; F 1,22 = 1.6, P = 0.21). Similarly, plasma corticosterone levels were higher in GRlo mice following 40 min restraint stress (Fig. 1C and D; area under the curve: F 1,22 = 12.9, P = 0.0017), consistent with previous data in GR+/ mice (Ridder et al., 2005). Elevated active-phase corticosterone levels in GRlo mice may reflect a ‘compensated’ state (Michailidou et al., 2008). In support of this, hippocampal expression of the GR-inducible target gene, GILZ, did not differ between GRlo and GRnorm mice (Table 1). While MRhi had no impact on stress-induced corticosterone levels, it abolished the effect of low levels of GR upon the corticosterone response to stress (main effect of MR: F 1,22 = 0.1, P = 0.76; MR-by-GR; F 1,22 = 6.1, P = 0.021, Fig. 1C and D). Overall, the data suggest that over-expression of MR ‘rescues’, in part, the inadequate feedback control after stress with GR deficiency. Thus, there is interaction between GR and MR in HPA axis feedback control after stress.

3.3. MR and GR independently affect cognition There was no effect of either MR or GR manipulation on cognitive performance in the Y-maze (Supplementary material, Table 1). Furthermore, there was no difference between genotypes in the ability to learn the location of a water maze platform over 8 days of trials (Fig. 2A) (day effect: F 4.8,156.6 = 49.8, P < 0.0001). On day 9, 24 h following the last trial, retention of memory was tested by removing the platform in a 1 min ‘‘probe’’ test. The dwell time in the target quadrant was increased in MRhi mice (F 1,35 = 14.7, P = 0.0005; Fig. 2B) as was the number of platform-site crossings (pin-point memory) (F 1,35 = 9.1, P = 0.005; Fig. 2C). However, while pin-point memory was unaffected by GR levels, time in the target quadrant was subject to an MR-by-GR interaction (F 1,35 = 9.0, P = 0.005) such that only when coupled with GR under-expression did MRhi increase dwell time (Bonferroni post hoc, P < 0.005). This suggests that elevated MR concomitantly with low GR levels may facilitate recall, or alternatively, could reflect reduced exploratory flexibility. To test whether high MR levels decrease ability to extinguish memory when circumstances alter, the hidden platform was switched to the opposite quadrant to that previously learned by the mice. All mice were able to learn the new location of the platform over the 4 days of testing (F 2.1,73.6 = 70.5, P < 0.0001, Fig. 3A). However, although MRhi mice and GRlo mice took longer to reach the platform in the new location (MR: F 1,35 = 13.8, P = 0.0007; GR: F 1,35 = 5.4, P = 0.027) there was no interaction between MR and GR (Fig. 3A). This appeared to reflect, in part, a difference in strategy since the percentage dwell time in the

Figure 1 Effect of MR/GR manipulations on plasma corticosterone levels (CORT). N = 8—10/genotype and values are mean  SEM. (a) Basal CORT levels (nmol/L) in the morning were unaffected by MR or GR manipulations. (b) Circadian peak CORT levels (nmol/L) in the evening were significantly higher in GRlo mice (*P < 0.05). (c) CORT response (nmol/L) to 40 min restraint stress. (d) Analysis of the area under the curve (nmol/L by time) reveals that GRlo, only when coupled with MRnorm, lead to a greater and longer CORT response (***P < 0.001, Bonferroni post hoc).

MR:GR balance and behaviour

Figure 2 Spatial learning and memory in the water maze, N = 8—11/genotype and values are mean  SEM. (a) Time taken (s) to find the platform decreased significantly over the days (P < 0.0001) and did not differ amongst the groups. Swim speed and distance to reach the platform did not differ (data not shown). (b) Dwell time (%) in the target quadrant during the probe trial was significantly higher in GRloMRhi mice relative to GRnormMRhi (post hoc tests, **P < 0.005, ns = not significant). (c) Number of platform crossings during the probe trial was significantly higher in MRhi mice, regardless of GR levels (**P < 0.005).

old goal site on the last day of testing of the new platform location was greater in MRhi mice (F 1,35 = 12.6, P = 0.001; Fig. 3B and C). As with the probe trial at the end of the original learning (Fig. 2B), there was a significant MR-by-GR

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Figure 3 Reversal learning in the water maze, N = 8—11/genotype and values are mean  SEM. (a) Time taken (s) to find the platform in its new location decreased significantly over the days (P < 0.0001). Irrespective of GR, MRhi mice were impaired in learning the new location (P < 0.001), and irrespective of MR, GRlo mice also showed impaired reversal learning (P < 0.05). (b) Time spent (%) in the old quadrant in swim 1 on day 4 of reversal testing. (c) A representative swim path from each group, from swim 1 day 4 of reversal learning, illustrates that GRloMRhi mice persisted in searching in the old goal site. Black circle represents old platform location; white circle represents new platform location.

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Figure 5 Fear memory formation and extinction in the passive avoidance test, N = 7—11/genotype and values are mean  SEM. Latency to transfer into the dark compartment (s) over test days. By day 6 GRnormMRnorm mice had significantly lower latencies compared with test day 4, and GRloMRnorm mice had significantly lower latencies by days 8—9 (Dunnett’s post hoc tests, significantly different to day 4, #P < 0.05). However, MRhi mice, irrespective of GR levels, showed no evidence of extinction throughout the experiment. Memory decay control mice (grey triangle) showed high latencies on days 7 and 11, despite a long interlude between the shock and test trial, thus memory for the shock does not decay over time.

Figure 4 Rigidity of response in the water maze N = 8—11/ genotype and values are mean  SEM. (a) Time taken (s) to mount the visible platform, when familiar spatial cues were present, was significantly higher in MRhi mice, irrespective of GR levels (***P < 0.0001). (b) One representative swim path from each group demonstrates that, despite the platform being highly visible, upon release most mice swam to the old goal site first and that MRhi mice continued to search in this old goal site for the majority of the trial (black circle represents old platform location; white circle represents visible platform). (c) Time taken (s) to mount the visible platform, with familiar spatial cues removed, did not differ significantly amongst the groups.

interaction. MRhi only increased dwell time when in concert with GRlo in the first swim on the last day of reversal learning (F 1,35 = 7.3, P = 0.010, Fig. 3B). To further investigate whether MR:GR balance is important in cognitive flexibility, mice were first trained to find a

hidden platform then given a trial with a clearly visible escape platform in the opposite quadrant. Again MRhi mice took longer than MRnorm mice to escape to the platform (F 1,34 = 23.4, P < 0.0001, Fig. 4A). There was no effect of GR levels, nor was there a significant interaction between MR and GR. The increased latency of MRhi mice to reach the visible platform in the new location was due to perseverant searching in the old platform site (Fig. 4B) and not visual or non-spatial motivational differences, since there was no effect of MRhi and/or GRlo on the latency to reach a visible platform in the absence of spatial cues (Fig. 4C). Thus, MRhi mice show impaired reversal of a learned spatial memory and when GR is concomitantly low, perseverance but not specific spatial memory, is reinforced‘.

3.4. Acquisition and extinction of fear memory is sensitive to GR and MR levels We next tested memory reversal in a different paradigm, albeit one involving the amygdala as well as the hippocampus. MR and GR levels influenced both formation and extinction of a fear-related memory in a passive avoidance test (latency to move from a light to a dark compartment). During the extinction trials, during which the mice did not receive a shock upon transfer to the dark compartment, both MRhi (F 1,31 = 15.4, P = 0.0005, Fig. 5) and GRlo mice (F 1,31 = 10.2, P = 0.0032) took significantly longer to transfer from the light to the dark compartment, but there was no significant MR-byGR interaction. MRhi mice, irrespective of GR levels, showed no reduction in latency to transfer to the dark compartment

MR:GR balance and behaviour during any of the extinction trials, thus high levels of MR associated with a significant delay in fear memory extinction. GRlo mice, regardless of MR levels, took longer to reduce their transfer latencies than did GRnorm mice, but by days 8—9 were transferring to the dark compartment faster than previously: evidence of fear memory extinction (Fig. 5). In sum, these data suggest that either GR haploinsufficiency or high expression of MR in forebrain independently limit extinction of non-spatial memory, with no interaction between the effects of low GR and high MR levels.

3.5. Anxiety and depressive-like behaviours are normal in MRhi and GRlo mice Interpretation of tests of fear-related and other memory functions may be confounded by genotype differences in affective function or pain threshold. However, there was no significant effect of MRhi or GRlo, either alone or in concert, on any parameters measured in the elevated plus maze, open field or light-dark box (Table 1, Supplementary material). Similarly, neither high MR nor low GR levels affected either pain threshold or behaviour in the tail suspension test, a test of depressive-like behaviour (Table 1, Supplementary material).

4. Discussion Our data illustrate the exquisite complexity of the stress system and emphasise the importance of context upon the behavioural and cognitive effects of altered MR:GR balance. MR and GR interact to control the HPA axis in response to restraint stress, notably when GR is low. Importantly, in the limbic-associated cognitive behaviours, altered levels of both GR and MR had independent and interactive consequences for the strategy used to reverse a learned spatial memory. Crucially, the failure to extinguish learned behaviours in MRhi mice prompts a reassessment of the conventional memory retention-enhancing effects mediated by this receptor (e.g. Ferguson and Sapolsky, 2008; Lai et al., 2007) suggesting that at high levels, MR engenders inflexible ‘fixed’ memories which might underlie a vulnerability to psychopathology, especially if GR is concomitantly low. Finally, as previously shown, changes in MR or GR did not induce compensatory changes in mRNA levels of the other receptor in the hippocampus or amygdala where they are most highly coexpressed (Lai et al., 2007; Wei et al., 2004). This contrasts with other transgenic models in which expression of one receptor causes compensatory changes in the other (e.g. Berger et al., 2006; Rozeboom et al., 2007). The nature of the transgene and its temporal expression as well as environmental conditions, including developmental ‘programming’, may determine if compensatory changes occur.

4.1. MR and GR interact to control restraint stress-induced HPA axis activation Since GILZ mRNA levels were not lower in GRlo mice, under basal circumstances, GRlo mice appear in a state of ‘balanced homeostasis’ with activation of the HPA axis compensating for reduced GR levels (Ridder et al., 2005; Tronche et al., 1999). In our experiment brains were taken between 12:00

655 and 15:00 before CORT levels begin to rise in anticipation of the dark phase in our control mice. However, it remains possible that under stressful conditions, reduced GR may cause HPA axis overshoot and higher plasma corticosterone levels earlier in the diurnal rhythm. High MR, which itself had no impact on basal glucocorticoid levels, minimised the HPA axis ‘‘stress’’ overshoot due to low GR levels, suggesting that under stressful conditions MR provides a negative feedback signal, particularly in a ‘‘glucocorticoid-resistant’’ state. Previously, elevated hippocampal MR capacity was associated with a hypoactive HPA axis response to stress in different strains of rats (Oitzl et al., 1995). This agrees with previous pharmacological findings that when GR levels are normal, antagonism or genetic deletion of hippocampal MR disinhibits negative feedback on the HPA axis under basal and peak conditions (Pace and Spencer, 2005; Ratka et al., 1989). Also, MR blockade enhances HPA axis response to stress, provided stressful information is processed by the hippocampus (Ratka et al., 1989). The HPA axis in GRlo mice may mimic the situation in major depression in which GR hypofunction is inferred from genetic and pharmacological studies of the sensitivity of the HPA axis to negative feedback by glucocorticoids (at brain and/or pituitary) (Holsboer, 2000). Since cyclical depressive episodes seem to follow episodes of elevated cortisol, maximising MR function — thus constraining HPA axis hyperactivity and its contribution to psychopathology — may represent an attractive strategy to treat major depression in some subsets of patients, especially as an increase in MR levels appears important early in antidepressant action (Seckl and Fink, 1992) and the evidence that enhanced activity of MR haplotype 2 is associated with optimism and protection against depression (Klok et al., 2011). Assessment by MR genotype (Klok et al., 2011) and/or pharmacological tests of combined GR and MR function (e.g. Mattsson et al., 2009) may be of use to stratify antidepressant therapy, targeting individuals with GR hypofunction and yet retained MR responsivity.

4.2. MR and GR interact to mediate aspects of ‘‘unlearning’’ With respect to cognition, both in stressful (water maze) and less stressful tasks (Y-maze) MRhi and GRlo mice often exhibited similar phenotypes so that any interaction between MR and GR genotypes (reflecting ‘imbalance’) would be unlikely to be manifest. These data are consistent with previously published data in which viral-mediated over-expression of MR with simultaneous under-expression of GR in the hippocampus had no impact on learning in the water maze (Ferguson and Sapolsky, 2008). However, in the water maze in the absence of an escape platform, MR and GR interacted to govern the search strategy. Thus, GRloMRhi mice mainly searched close to where the platform was previously (a rigid strategy), whereas GRnormMRhi mice mainly searched away from the original location after they had discovered the platform absent (a more flexible and explorative strategy). A role of hippocampal MR in appraisal and strategy switching in novel situations has previously been documented (e.g. Schwabe et al., 2010); our data show an important role of GR levels in modulating this.

656

4.3. MRhi and GRlo independently impair reversal learning Overly persistent searching for an absent platform, as seen in the MRhi mice, may be indicative of a perseverant phenotype. Indeed, previously trained MRhi mice persisted in searching for the removed platform, failing to escape to a visible platform in the opposite quadrant (though they escaped readily when spatial cues were removed), and were slow to learn the new location during the reversal learning trials. Intriguingly, reduced GR levels, irrespective of MR levels, also impaired reversal learning. However, both manipulations (MRhi and GRlo) effectively result in a higher MR:GR ratio, which may provide unfavourable conditions in the brain for effective reversal learning. However, since GRloMRhi mice were similarly impaired, these data suggest that absolute levels (i.e. high MR, regardless of whether GR levels were normal or low) rather than the relative ‘balance’ between the receptors determine reversal learning ability. Naturally, glucocorticoid signalling is affected by many more factors (e.g. pre-receptor metabolism, corticosterone binding globulin activity) but it is clear that merely variation in receptor density can have profound effects. The perseverant phenotype of MRhi or GRlo mice was also present during fear memory testing. Glucocorticoids are critical for fear memory extinction in humans and rodents (Brinks et al., 2009; De Quervain et al., 2011). Glucocorticoids administered immediately after a trauma (e.g. electric shock) enhance memory consolidation in rodents, but if given immediately prior to re-test, glucocorticoids enhance extinction (Joels et al., 2006). GR activation facilitates extinction in the active avoidance response but inhibits the retention of a passive avoidance response (Kovacs et al., 1976, 1977). MR antagonism impairs acquisition and retrieval of fear motivated behaviour (Zhou et al., 2011), while MR agonism impairs memory extinction during contextual fear conditioning in rats (Ninomiya et al., 2010). These functions suggest distinct noninteractive roles of the two receptors in fear memory extinction and highlight that the timing of glucocorticoid administration and the nature of the test is important. Although the exact mechanism by which MR or GR influence extinction learning is unclear, glutamate receptors (NMDA, metabotropic glutamate and AMPA receptors), GABAergic receptors and brain derived neurotrophic factor (BDNF) play a role in extinction learning (Myers et al., 2011; Pang et al., 2011; Soliman et al., 2010). Glucocorticoids regulate all of these signalling systems (e.g. Krugers et al., 2010; Weiland et al., 1997). Whatever the molecular processes, the impaired extinction/reversal learning, especially prominent in MRhi mice, may reflect a cognitive inflexibility, a lack of switching between memory systems and persistence of a particular memory that might underlie vulnerability to affective disorders such as PTSD. In support, MRs are critical for the expression of cognitive flexibility during stress (i.e. switching between appropriate behavioural strategies) (Oitzl and de Kloet, 1992; Schwabe et al., 2010). However, it is possible that an increased delay in entering the dark compartment in the passive avoidance test results from increased appraisal time. And it is currently unclear whether, all else being equal, a ‘stronger’ memory takes longer to extinguish than a weaker memory. This will be the subject of future

A.P. Harris et al. studies. Nevertheless, increased limbic MR, perhaps as a result of early life stress (Llorente et al., 2011) or genetic variation (van Leeuwen et al., 2011), may set up vulnerability to subsequent adult psychopathology. This suggests the possibility that MR blockade as well as GR agonism may be a potential prevention or treatment strategy of PTSD in vulnerable individuals after traumatisation.

4.4. No impact of MRhi or GRlo on affective behaviour The affective impacts of MR and GR manipulations seen in some studies (Rozeboom et al., 2007; Tronche et al., 1999), and indeed in our own previous study (Lai et al., 2007), were not found here, mirroring other previous work (Berger et al., 2006; Oitzl et al., 2001; Ridder et al., 2005). The discordance, particularly with our previous data, is likely due to differences in environmental conditions, notably parental influences and housing conditions. Specifically, group housing (with mixed genotypes) was used here, whereas mice were singly housed (deemed stressful) in our previous study (Lai et al., 2007). Chronic or acute stress, engender anxious or depressive-like phenotypes in GR+/ mice (Ridder et al., 2005), with prominent effects of single housing in the C57Bl/6J background strain (Kulesskaya et al., 2011), and may similarly alter behaviour of MRhi mice. Maternal influences could also be a factor. In contrast to previous studies where wild-type females were bred with MRhi males, here, to maintain the same maternal environment for all four genotypes, we exclusively crossed MRhi females with GRlo males. Both maternal and paternal ‘early life’ contributions (intrauterine, post-natal care and/or epigenetic factors) determine vulnerability to affective disorders (Liu et al., 1997). Indeed, it has recently been reported that on a C57BL/6 background, GR+/ dams provide less maternal care (licking and grooming) than wild-type dams (Chourbaji et al., 2011). Thus MR/GR influences on maternal behaviour are plausible and may contribute to the discordance between studies in this domain. Indeed much literature suggests that affective phenotype is subject to developmental programming, notably by glucocorticoid/stress exposure (e.g. Holmes et al., 2006). Nonetheless, in littermates, GR and MR manipulations impact upon cognition suggesting it is less sensitive to early life and adult environmental effects. At which stage this effect arises remains to be determined. Finally, the degree of manipulation (e.g. total knock out versus reduced expression) may account for some of the incongruous findings on the effects of MR/GR manipulations on affective phenotype (e.g. Tronche et al., 1999). It should also be noted that central MR can influence blood pressure (e.g. Gomez-Sanchez and Gomez-Sanchez, 2012), which may confound behavioural and/or HPA axis responses. However, these MR-mediated effects are mainly thought to reflect actions on the hindbrain outside the distribution of expression of the transgene (the hypothalamus which forms a reciprocal barostatic circuit with the brainstem is also outside the expression of the transgene) and we found no impact of forebrain MR overexpression on resting blood pressure (Lai et al., 2007). It is, however, unclear if blood pressure under stress is altered in these animals. In summary, we show that the MR:GR balance hypothesis explains specific aspects of HPA axis feedback under stress

MR:GR balance and behaviour and spatial memory, but that other HPA and cognitive effects are specific to absolute MR and/or GR levels without interaction. When imbalance occurs it plausibly underpins pathogenesis. Manipulations of MR in particular deserve exploration in psychotherapeutics.

Role of the funding sources This work was supported by a European Science Foundation (EuroSTRESS) grant to APH, JRS and ERK.

Conflict of interest E.R. de Kloet is an advisor to Dynacorts Therapeutics and Corcept Therapeutics, though he has no commercial interest in the material presented in this manuscript. All other authors declare that they have no conflicts of interest.

Acknowledgements This work was supported by a European Science Foundation (EuroSTRESS) grant to APH, JRS and ERK. We would like to thank three anonymous reviewers for their constructive comments on the manuscript.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.psyneuen.2012.08.007.

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