Metaplasticity of amygdalar responses to the stresshormone corticosteroneHenk Karsta,b,1, Stefan Bergerc, Gitta Erdmannc, Günther Schützc, and Marian Joëlsa,b

aDepartment of Neuroscience and Pharmacology, University Medical Center Utrecht, 3508 AB, Utrecht, The Netherlands; bSwammerdam Institute for LifeSciences–Center for Neuroscience, University of Amsterdam, 1098 XH, Amsterdam, The Netherlands; and cGerman Cancer Research Center, 69120 Heidelberg,Germany

Edited* by Donald W. Pfaff, The Rockefeller University, New York, NY, and approved June 24, 2010 (received for review December 15, 2009)

High levels of corticosteroids (as circulate after stress) quickly andreversibly enhance hippocampal glutamatergic transmission via non-genomicactions requiringmineralocorticoid receptors. Subsequently,the hormone slowly and long-lastingly normalizes hippocampal cellfunction, through nuclear glucocorticoid receptors. Here we describea rapid mineralocorticoid receptor-dependent enhancement of glu-tamatergic transmission in basolateral amygdala neurons. Contraryto the hippocampus, this rapid enhancement is long-lasting, poten-tially allowing an extended window for encoding of emotionalaspects during stressful events. Importantly, the long-lasting changein state of amygdala neurons greatly affects the responsiveness tosubsequent surgesof corticosterone, revealingaquick suppressionofglutamatergic transmission, which requires the glucocorticoid re-ceptor. Responses of basolateral amygdala neurons to the stresshormonecorticosteronecan thus switch fromexcitatory to inhibitory,depending on the recent stress history of the organism.

basolateral amygdala | glucocorticoid receptor | glutamate | miniatureexcitatory postsynaptic current | mineralocorticoid receptor

Shortly after stress, corticosteroid hormones are released inhigh amounts from the adrenal glands, enter the brain, and

predominantly bind to intracellular glucocorticoid receptors(GRs), e.g., in the hippocampal CA1 area (1, 2). Neurons in thisarea also highly express intracellular mineralocorticoid receptors(MRs), but because of their high affinity these receptor are al-ready largely occupied even under rest. Both receptor types actas transcription factors, altering the expression of responsivegenes (3). In this way corticosterone (the prevailing hormone inrodents) changes cellular excitability in the CA1 area, starting 1–2 h after an elevation in corticosteroid level and lasting up to atleast several hours (4). Overall, MRs serve to maintain gluta-matergic transmission and viability in the CA1 area, whereasGRs cause a delayed suppression of synaptic transmission andplasticity, presumably normalizing stress-dependent rises in ac-tivity (4). Thus, the two receptor types exert a slow genomic yin–yang control over hippocampal excitability.Recently, though, we demonstrated that, in the CA1 area,

corticosterone also quickly and reversibly enhances the frequencyof miniature excitatory postsynaptic currents (mEPSCs), each ofwhich reflects the spontaneous release of a glutamate-containingvesicle (5). This nongenomic effect critically depends on thepresence of MRs thought to reside in the presynaptic terminalmembrane, and involves activation of the ERK1/2 pathway (6).The apparent affinity of this membrane-located MR is 10-foldlower than that of the intracellular MR, allowing it to play aprominent role in the behavioral stress response (7). It is thoughtthat corticosterone via membrane MRs (in close interaction withother stress mediators such as noradrenaline and CRH) quicklyraises hippocampal excitability, to be normalized 1–2 h laterthrough gene-mediated GR actions (8). This leaves a restrictedtime-window during which the encoding of stress-related in-formation is facilitated (2, 9, 10).Exposure to stressful situations, however, will nearly always

also activate the amygdala nuclei (10–12). Recent studies suggest

that slow GR effects in the basolateral amygdala (BLA) enhancerather than suppress excitatory transmission (13, 14), indicatingthat corticosterone via its genomic pathway affects CA1 andBLA neurons differently. We wondered whether this is also truefor rapid nongenomic effects of corticosterone. Rapid effects areindeed expected to take place, because corticosteroid receptorswere observed in the membrane of LA neuron terminals withimmunogold electron microscopy (15).

ResultsCoronal slices containing the amygdala nuclei were prepared frommale young-adult C57/Bl6 mice under rest in the morning, i.e.,when corticosteroid levels are very low, as described in ref. 14.Application of 100 nM corticosterone (for 20 min) in vitro in-creased the mEPSC frequency of principal neurons in the BLA butnot the central amygdala (Fig. 1 A–C and E). The mEPSC am-plitude in both areas was unaffected (Fig. 1 D, F, and H), as wasthe mEPSC τ of decay in the BLA (baseline, 8.2 ± 0.7 ms; duringcorticosterone, 11.9 ± 1.8 ms; n = 7; paired t test, P = 0.063) andthe central amygdala (baseline, 15.3 ± 1.7 ms; during corticoste-rone, 19.2 ± 2.1 ms; n= 9; P= 0.074), although a tendency towardan increase was observed in both areas. Our first conclusion is that,in the BLA, corticosterone induces comparable rapid-onset effectson the mEPSC frequency as in the hippocampal CA1 area but thiscannot be generalized to all limbic brain nuclei.The cumulative histogram (Fig. 1G) suggests that rapid effects

of corticosterone on BLA mEPSC frequency are not quickly re-versible, in contrast to what was earlier seen in the hippocampalCA1 area. In a subset of cells that were recorded up to 20 minafter washout of corticosterone, mEPSC frequency indeed re-mained high (baseline, 3.2 ± 0.4 Hz; ∼20 min after corticosteronewash-out, 4.0 ± 0.2 Hz; n = 5, P = 0.02 with a paired t test).Because it is difficult to examine effects of corticosterone overeven longer periods of time with whole-cell patch-clamping ofadult neurons, we compared (in separate groups of cells) mEPSCfrequency several hours after a brief pulse of corticosterone withthat after vehicle treatment, both in hippocampal CA1 and BLAneurons. A brief pulse of corticosterone (100 nM for 20 min) wasindeed sufficient to cause high mEPSC frequency several hourslater in principal neurons of the BLA [corticosteroid (n = 7)versus vehicle-treated cells (n = 7), P = 0.002, unpaired t test] butnot the CA1 area (n = 10 and 5 cells respectively, P > 0.05; formean values of corticosterone-treated cells, see Fig. 2 A and B).To determine the involvement of the two known corticosteroid

receptor types in these effects, we made use of male mutant micewith forebrain ablation of either MR or GR induced at the age of

Author contributions: H.K. and M.J. designed research; H.K. performed research; S.B.,G.E., and G.S. contributed new reagents/analytic tools; S.B., G.E., and G.S. generatedunique mice; H.K. and M.J. analyzed data; and H.K. and M.J. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1To whom correspondence should be addressed. E-mail: h.karst@umcutrecht.nl.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.0914381107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0914381107 PNAS | August 10, 2010 | vol. 107 | no. 32 | 14449–14454

NEU

ROSC

IENCE

8–9 wk [MRCaMKCreERT2 and GRCaMKCreERT2, respectively (16);SI Methods] and their respective littermate controls. Cortico-steroid effects were clearly visible in GRCaMKCreERT2 mice (Fig.2 C and D), which do not express GRs but do have MRs in theBLA. By contrast, in MRCaMKCreERT2 mice [i.e., in the absenceof BLA MRs (SI Methods)] rapid enhancement of the mEPSCfrequency was no longer observed (Fig. 2 E and F), supportinga critical role of MRs in the rapid development of raised mEPSCfrequency by corticosterone. Enhanced mEPSC frequency in thepresence of corticosterone was observed in all cells (n = 5) fromcontrol littermates (MRflox/flox or GRflox/flox), to an extent com-parable to that seen in control C57/Bl6 mice (mean frequency ±SEM before corticosterone, 2.28 ± 0.24 Hz; during CORT,3.84 ± 0.84 Hz). The findings in genetically modified mice wereconfirmed by pharmacological observations in control C57/Bl6animals. Thus, selective activation of MRs by application of(10 nM) aldosterone in the presence of a GR antagonist (500 nMRU 38486) resulted in strong enhancement of the mEPSC fre-quency (Fig. 2G). Conversely, application of the selective GR

agonist RU 28362 (100 nM) did not enhance but, rather, de-creased the mEPSC frequency (Fig. 2G).Gradual enhancement of mEPSC frequency was not present

after corticosterone administration in MRCaMKCreERT2 mutants(Fig. 2F), nor after RU 28362 application (Fig. 2G), emphasizingthat MRs are necessary for the development of both the rapidand lasting enhancement in mEPSC frequency. Interestingly, inthe GRCaMKCreERT2 mice (where corticosterone selectively acti-vates MRs) mEPSC frequency did not remain at a high level butreturned to baseline values, as is evident from cells recorded 1–4 hafter a 20-min pulse of corticosterone (Fig. 2D). In the samevein, application of aldosterone in the presence of RU 38486induced only a transient response (Fig. 2G). Transient en-hancement in mEPSC frequency was also observed when corti-costerone was applied in the presence of the protein synthesisinhibitor cycloheximide (Fig. 2G). This suggests that MRs maybe necessary but are certainly not sufficient for the maintenanceof the high mEPSC frequency; the sustained effect also requiresa protein synthesis-dependent process via the GR.

A B

C D

E F

G H

Fig. 1. Corticosterone enhances mEPSC frequency of BLAbut not CeA principal neurons, as shown for typical traces inA and B, respectively. The mEPSC frequency over time isshown for BLA neurons (average of n = 7 cells) before andduring application of 100 nM corticosterone in C. Comparisonof the averaged frequency during baseline (t = −5–0 min) andthe final 5 min during corticosterone administration (t = 15–20 min) revealed a statistically significant difference (paired ttest, *P < 0.005). No change was observed during cortico-sterone administration on mEPSC amplitude (D). In the CeA,neither mEPSC frequency (E; average of seven cells) nor am-plitude (F) was affected by corticosterone administration.The cumulative distribution of mEPSC frequencies in BLAneurons (G) showed a significant enhancement during corti-costerone administration (solid line; Kolmogorov–Smirnovanalysis, P < 0.005) compared with baseline (dashed line),which did not reverse 20 min after washout (dotted line). Nochange was observed in the frequency distribution of mEPSCamplitudes during compared with before corticosterone ap-plication (H; baseline in thin bars, the situation during corti-costerone application in bold; dotted and solid linesrepresent the Gaussian fits before and during corticosteroneapplication, respectively).

14450 | www.pnas.org/cgi/doi/10.1073/pnas.0914381107 Karst et al.

A B

C D

E F

G

Fig. 2. MR and GR requirement for the development of rapid and delayed corticosteroid effects in the BLA. Application of corticosterone rapidly enhancedmEPSC frequency in CA1 pyramidal neurons (A; black bar, n = 5) compared with pretreatment baseline frequency (open bar; paired t test, P < 0.05). Neuronsrecorded 1–4 h after slices were treated for 20 min with 100 nM corticosterone displayed a low mEPSC frequency (light gray bar, n = 10). In the BLA, cor-ticosterone treatment also quickly enhanced mEPSC frequency (B; n = 7, P < 0.005). However, 1–4 h after slices were treated for 20 min with 100 nM cor-ticosterone, mEPSC frequency was still elevated (n = 7, see main text). In mice with induced forebrain ablation of GR (GRCaMKCreERT2), application ofcorticosterone also resulted in rapid enhancement of mEPSC frequency (C and D; n = 8 and 5 cells, respectively) in the BLA. This enhancement, though, wastransient (D; n = 4 cells, for recordings 1–4 h after corticosterone application). In mice with induced forebrain ablation of MR (MRCaMKCreERT2), neither rapid(n = 7) nor delayed (n = 6) effects of corticosterone were observed (E and F). Corticosterone in the presence of a protein synthesis inhibitor (cycloheximide)was still able to induce an increase in mEPSC frequency (n = 7, P = 0.03), but the effect was only transient (G). Selective activation of the MR (by aldosteronein the presence of RU 38486; n = 7, P = 0.004) briefly enhanced mEPSC frequency, in contrast to the GR-agonist RU 28362, which even reduced the frequency(n = 5, P = 0.03). Data are expressed as the mean (±SEM) change in mEPSC frequency during the final 5 min of drug application (black bar for each drug) or20 min after washout (gray bar), in both cases relative to the average frequency at t = −5–0 min (just before drug application, set at 100%). *P < 0.05compared with baseline (paired t test).

Karst et al. PNAS | August 10, 2010 | vol. 107 | no. 32 | 14451

NEU

ROSC

IENCE

A

C

B

D

E

Fig. 3. Exposure to two pulses of corticosterone (10- to 20-min pulse duration, 1- to 3-h pulse interval) induced comparable elevations (P < 0.05) in mEPSCfrequency in CA1 pyramidal cells (A; mean ± SEM of n = 6 and 5 cells during the first and second pulse, respectively). By contrast, BLA neurons responded to thefirst pulse with increased mEPSC frequency (n = 12, P < 0.05), but with a decreased (P < 0.05) mEPSC frequency to a second pulse (n = 6). In BLA cells from animalsexposed to restraint stress before slice preparation, corticosterone also rapidly decreased mEPSC frequency (B; n = 8 cells). (C) A significant (P < 0.001) inverserelationship was observed between baseline mEPSC frequency and the percentual change in mEPSC frequency during corticosterone treatment (compared withbaseline; no change = 0). For this figure, data obtained in mice with low corticosterone level (under rest, at the circadian nadir) were combined with thoseobtained 1–4 h after treatment with corticosterone in vitro or restraint stress (in all cases C57/Bl6). Correlation was tested for significance with a Pearson test. (D) InGRflox/flox controls, we observed a significant reduction in mEPSC frequency after stress (n = 9 cells, P < 0.05), similar to what we observed in wild-type C57/Bl6 mice(n = 8). The reduction was not observed in GRCaMKCreERT2 mutants (n = 6 cells). (E) The protein synthesis inhibitor cycloheximide did not interfere with thesuppressing effect of corticosterone in slices from stressed mice (n = 5, P = 0.04). The GR-agonist RU 28362 decreased mEPSC frequency after stress (n = 5, P = 0.04),whereas selective activation of the MR (by aldosterone in the presence of RU 38486; n = 6) did not change the frequency. If corticosterone was applied in thepresence of the endocannabinoid-R1 antagonist SR141716A (1 μM), mEPSC frequency was not significantly altered (n = 6). Data are expressed as the mean (+SEM)change in mEPSC frequency during the final 5 min of drug application (black bar for each drug) or 20 min after washout (gray bar), relative to the frequency at t =−5–0 min (just before drug application, set at 100%). *P < 0.05 compared with baseline (paired t test).

14452 | www.pnas.org/cgi/doi/10.1073/pnas.0914381107 Karst et al.

Because one pulse of corticosterone apparently causes pro-longed enhancement of mEPSC frequency in BLA neurons, onemay wonder whether this makes BLA as opposed to CA1 hip-pocampal cells refractory to a second pulse, hours later. In thehippocampal CA1 area, mEPSC frequency several hours aftercorticosterone exposure was low (Fig. 2A), and cells respondedto renewed corticosterone exposure with enhanced mEPSCfrequency, to a comparable extent as after the first pulse (Fig.3A). Surprisingly, though, renewed exposure of BLA neurons tocorticosterone (in cells that still exhibited increased mEPSCfrequency due to the first pulse; Fig. 2B) caused a decrease inmEPSC frequency (Fig. 3A). Like the increase in mEPSC fre-quency seen after the first pulse, the decrease in response toa second pulse was long lasting; thus, >1 h after washout of thesecond corticosterone application, mEPSC frequency was ata comparably low level (0.88 ± 0.11 Hz) as seen at the end of thesecond pulse (Fig. 3A).We probed the physiological significance of the reduced

mEPSC frequency in response to a second corticosterone ap-plication, by exposing mice to restraint stress 20 min beforepreparing slices. In accordance with the lasting enhancement inmEPSC frequency observed after in vitro administration ofcorticosterone, the baseline mEPSC frequency of BLA neuronsin slices from stressed animals was high (∼4 Hz; Fig. 3 B and D).Under these conditions, neurons responded to corticosteroneexposure in vitro with a rapid and lasting decrease in mEPSCfrequency (Fig. 3 B and D). Thus, in slices prepared from animalsunder rest, corticosterone long lastingly increases mEPSC fre-quency; once the frequency is enhanced (e.g., by stress), sub-sequent corticosterone exposure decreases mEPSC frequency. Infact, the mEPSC frequency before corticosterone applicationpredicted the nature of the rapid hormone effects, showinga clear inverse relationship (B = −0.17, P < 0.001; Fig. 3C). Acomparable inverse relationship (B = −0.21, P < 0.05) was alsoobserved for a limited number of cells (n= 6) tested in vitro withthe membrane-impermeable ligand CORT-BSA.The rapid decrease in mEPSC frequency was recapitulated in

restrained GRflox/flox controls but not in BLA neurons of re-strained GRCaMKCreERT2 mice (Fig. 3D), indicating that therapid decrease critically depends on the local presence of GRs.This was confirmed by experiments in control C57/Bl6 micewhere the selective GR agonist RU 28362 significantly decreasedmEPSC frequency in BLA neurons recorded after stress (Fig.3E); mEPSC frequency recorded 20 min after washout of RU28362 still tended to be low, but these values were more variable.Application of aldosterone in the presence of RU 38486 in slicesprepared from stressed animals did not change the mEPSCfrequency (Fig. 3E). The nongenomic nature of the mEPSCsuppression by corticosterone after stress was supported by theobservation that the response persisted in the presence of cy-cloheximide (Fig. 3E).Interestingly, if corticosterone was applied to slices from ear-

lier stressed C57/Bl6 mice but now in the presence of theendocannabinoid-R1 antagonist SR141716A (1 μM), we also didnot observe a decrease in mEPSC frequency but instead revealeda slight but nonsignificant enhancement (Fig. 3E). This suggeststhat the rapid suppressive and GR-dependent effects involveendocannabinoids, as was earlier reported for the hypothalamicparaventricular nucleus (17); this differs from the rapid MR-dependent actions observed in the hippocampus (and possiblyalso in the BLA), which do not seem to depend on retrogradesignaling (6). The current data with endocannabinoids alsoprovides a cellular basis for recent behavioral observations im-plicating endocannabinoids in amygdala-dependent memoryformation (18).

DiscussionWe here demonstrate that, in BLA neurons of an animal withlow circulating corticosteroid levels (i.e., under rest, at the cir-cadian nadir), temporary rises in hormone level—such as mayoccur during and some time after stress—cause a rapid-onsetenhancement of spontaneous glutamate-mediated activity viaMRs, similar to what was earlier described for CA1 hippocampalcells (5). In contrast to the transient effect in the CA1 area,though, the enhanced spontaneous glutamate-mediated activityin the BLA is maintained for several hours. For long-lastingeffects to occur, the development of rapid effects via MR isnecessary, but GR expression is also required for maintaininghigh mEPSC frequency. These findings suggest that rapid MR-dependent effects are functionally linked to later-developinggene-dependent and GR-requiring processes, along the sameline as rapid effects of estrogens were found to influence sub-sequently developing genomic actions (19). The fact that in-creased mEPSC frequency in BLA is sustained, as opposed tothe transient effects earlier reported for CA1 cells, fits with theslowly developing excitatory effects exerted in the BLA (butnot CA1) by corticosteroids on firing frequency accommodation(13, 14). The collective data support that, in the BLA (as op-posed to the CA1 area), excitability is raised by corticosteroneover a relatively long time-window. One could speculate that thiswould allow encoding of information via the amygdala over anextended period after stress, a mechanism that might contributeto the preferential facilitation by stress of retention of emotionalversus neutral information (10, 20).Unexpectedly, exposure to a rise in corticosteroid level against

a history of stress produced a rapid effect in the opposite di-rection, via a GR-dependent process. Apparently, the rapid non-genomic effects via MRs and GRs in the BLA serve a similaryin–yang function on cellular excitability as earlier postulated forthe genomic actions in the CA1 area, although in a differenttime-domain. Apparently, one hormone can induce opposite ef-fects, depending on the recent history of the organism and itsneurons. Interestingly, in BLA tissue prepared from animalsunder rest, the enhancement in mEPSC frequency prevails whenthe endogenous ligand corticosterone is applied, but suppressiveeffects can still be uncovered by selective activation of GR. Bycontrast, application of aldosterone/RU 38486 to slices fromstressed animals (or corticosterone to slices from stressedGRCaMKCreERT2 mice) did not lead to enhanced mEPSC fre-quency. These findings, combined with those earlier reported forthe hippocampus (6) and PVN (17), suggest that under rest themembrane of presynaptic terminals in the BLA is enriched withMRs, whereas only a limited number of GRs are present post-synaptically. After stress, MRs may internalize while the surfaceexpression of GRs seems enhanced. Electron-microscopicexperiments need to verify this.The "state-dependency" of rapid corticosteroid effects in the

BLA is reminiscent of the metaplasticity and sliding-thresholdearlier described with regard to synaptic plasticity in the hippo-campus after high-frequency stimulation (21). The functionalimplication could be that in the BLA, renewed exposure tocorticosterone some hours after stress helps to reset the spon-taneous glutamate transmission, earlier raised via corticosteroidsin interaction with other stress mediators like noradrenaline. Ifso, insufficient levels of corticosterone—as have been hypothe-sized to occur in individuals vulnerable to posttraumatic stressdisorder (22)—might fail to reset the BLA excitability, so thatpotentially ongoing encoding is not curtailed.

MethodsIn the current study, we usedmale young-adult C57/Bl6mice (6–9wk old; HarlanCPB), decapitated under rest in the morning (i.e., when corticosteroid levels arevery low) as described in ref. 14. In some experiments, animals were subjected torestraint stress for 20 min immediately before decapitation.

Karst et al. PNAS | August 10, 2010 | vol. 107 | no. 32 | 14453

NEU

ROSC

IENCE

Immediately after decapitation, the brain was removed from the skull andchilled (4 °C) in artificial cerebrospinal fluid (aCSF) containing 120 mmol/LNaCl, 3.5 mmol/L KCl, 5.0 mmol/L MgSO4, 1.25 mmol/L NaH2PO4, 0.2 mmol/LCaCl2, 10 mmol/L D-glucose, and 25.0 mmol/L NaHCO3 (gassed with 95% O2

and 5% CO2). Coronal slices (350 μm thick) containing the BLA and/or dorsalhippocampus were prepared with a vibroslicer (Leica VT 1000S). Slices werestored at room temperature until use in recording aCSF containing 120mmol/L NaCl, 3.5 mmol/L KCl, 1.3 mmol/L MgSO4, 1.25 mmol/L NaH2PO4, 2.5mmol/L CaCl2, 10 mmol/L D-glucose, and 25 mmol/L NaHCO3.

One slice at a time was placed in a recording chamber mounted on anupright microscope (Axioskop 2 FS plus; Zeiss) with differential interferencecontrast, water-immersion objective (63×), and 10× ocular. The slices werecontinuously perfused with aCSF (flow rate 2–3 mL/min, temperature 32 °C, pH7.4) consisting of 120 mM NaCl, 3.5 mM KCl, 1.3 mM MgCl2, 2.5 mM CaCl2, 25mM NaHCO3, 1.25 mM KH2PO4, and 10 mM D-glucose; bicuculline meth-chloride (20 μm; Tocris) and tetrodotoxin (0.5 μm; Latoxan) were added toblock GABAA receptor-mediated signals and action potentials, respectively.Whole-cell voltage-clamp recordings were made with an Axopatch 200B am-plifier (Axon Instruments) using borosilicate glass electrodes (impedance 4–6MΩ, 1.5-mm outer diameter; Hilgenberg) pulled with a micropipette puller(Brown/Flaming P-87; Sutter Instruments). For mEPSC recordings, the intra-cellular pipette solution contained 120 mM Cs methane sulphonate, 17.5 mMCsCl, 10 mM Hepes, 5 mM BAPTA, 2 mM MgATP, and 0.1 mM Na GTP (295mOsm, pH 7.4 adjusted with CsOH). BAPTA was obtained from MolecularProbes; all other chemicals were purchased from Sigma.

Neurons in the BLA were selected for recording if they displayed a pyra-midal-shaped cell body. All mEPSCs were recorded with a holding potentialof −70 mV. If the neuron under study displayed stable mEPSC propertiesduring baseline recording (at least 10 min), corticosteroids were appliedfor ≈20 min via the perfusion medium; drug testing comprised (i) 100 nMcorticosterone (Sigma), (ii) 10 nM aldosterone (Fluka) in the presence of500 nM RU 38486 (Sigma), (iii) 100 nM RU 28362 (generous gift from BennoRoozendaal, University Medical Center Groningen, Groningen, The Nether-lands), (iv) 100 nM corticosterone in the presence of 100 μM cycloheximide(Fluka), and (v) 100 nM corticosterone-BSA (Sigma). In one set of cells, wecompared cells 1–4 h after a 20-min application of corticosterone or vehicle,as described in ref. 14. Corticosterone, aldosterone, and RU28362 wereprepared weekly in a stock solution (1 mM in 95% ethanol) and diluted to

their final concentration in aCSF just before application. Cycloheximide andcorticosterone-BSA were dissolved directly in ACSF and freshly prepared onthe day of the experiment. RU38486 was dissolved in distilled water (1 mM)with a few drops of 5 M HCl and diluted to the final concentration in ACSF.

Series resistance compensation was >70% in all recordings. Series re-sistance and capacitance were monitored during the whole recording.Responses were filtered at 5 kHz and digitized at 10 kHz (Digidata 1322A;Axon Instruments). All data were acquired, stored, and analyzed on a PCusing pClamp 9.0 and Clampfit 9.2 (Axon Instruments). Minimal cutoff formEPSC analysis was 6 pA.

Mutant Mice. To generate mutant mice with forebrain ablation of either MRor GRwe crossed transgenic mice expressing the CreERT2 fusion protein underthe control of the regulatory elements of the CaMKIIa gene (16) to mice har-boring either a conditional MR allele (23) or a conditional GR allele (24). Eight-to 9-wk-old MRCaMKCreERT2 (MRflox/floxCaMKCreERT2) and their littermate con-trols (MRflox/flox), as well as GRCaMKCreERT2 (GRflox/floxCaMKCreERT2) and theirlittermate controls (GRflox/flox), were treated with 1 mg of tamoxifen twicea day for 5 d to induce in mutants the loss of MR or GR in forebrain neuronsthat express CreERT2, including principal neurons in the BLA (Fig. S1).

Statistical Analyses. In most cases, we applied paired t test comparisons be-tween mEPSC properties determined during the final 5 min of baseline re-cording and the final 5 min of recording in the presence of corticosterone [orother (combinations of) drugs] in the same cells. If mEPSC properties de-termined 1–4 h after corticosterone treatment were compared with thoseafter vehicle treatment, we used unpaired statistics. Cumulative frequencydistributions were subjected to a Kolmogorov–Smirnov analysis. We re-moved one observation (a cell tested with corticosterone in the presence ofa CB-R1 antagonist) from the dataset, because this value was >2 SD removedfrom the mean and considered to be an outlier.

ACKNOWLEDGMENTS. This work was supported by Dutch Organization forScientific Research (NWO-ALW) Grant 817.02.017 (to H.K.), as well as by theDeutsche Forschungsgemeinschaft through SFB 636 and EuropeanUnionGrantLSHM-CT-2005-018562 (CRESCENDO).

1. de Kloet ER, Joëls M, Holsboer F (2005) Stress and the brain: from adaptation todisease. Nat Neurosci Rev 6:463–475.

2. Ulrich-Lai YM, Herman JP (2009) Neural regulation of endocrine and autonomic stressrepsonses. Nat Neurosci Rev 10:397–409.

3. Lu NZ, et al. (2006) International Union of Pharmacology. LXV. The pharmacology andclassification of the nuclear receptor superfamily: Glucocorticoid, mineralocorticoid,progesterone, and androgen receptors. Pharmacol Rev 58:782–797.

4. Joëls M, Karst H, Krugers HJ, Lucassen PJ (2007) Chronic stress: Implications forneuronal morphology, function and neurogenesis. Front Neuroendocrinol 28:72–96.

5. Karst H, et al. (2005) Mineralocorticoid receptors are indispensable for nongenomicmodulation of hippocampal glutamate transmission by corticosterone. Proc Natl AcadSci USA 102:19204–19207.

6. Olijslagers JE, et al. (2008) Rapid changes in hippocampal CA1 pyramidal cell functionvia pre- as well as postsynaptic membrane mineralocorticoid receptors. Eur J Neurosci27:2542–2550.

7. Joëls M, Karst H, DeRijk R, de Kloet ER (2008) The coming out of the brainmineralocorticoid receptor. Trends Neurosci 31:1–7.

8. Joëls M, Baram TZ (2009) The neuro-symphony of stress. Nat Neurosci Rev 10:459–466.9. Joëls M, Pu Z, Wiegert O, Oitzl MS, Krugers HJ (2006) Learning under stress: How does

it work? Trends Cogn Sci 10:152–158.10. Roozendaal B, McEwen BS, Chattarji S (2009) Stress, memory and the amygdala. Nat

Neurosci Rev 10:423–433.11. McGaugh JL (2004) The amygdala modulates the consolidation of memories of

emotionally arousing experiences. Annu Rev Neurosci 27:1–28.12. Rodrigues SM, LeDoux JE, Sapolsky RM (2009) The influence of stress hormones on

fear circuitry. Annu Rev Neurosci 32:289–313.13. Duvarci S, Paré D (2007) Glucocorticoids enhance the excitability of principal

basolateral amygdala neurons. J Neurosci 27:4482–4491.

14. Liebmann L, et al. (2008) Differential effects of corticosterone on the slow

afterhyperpolarization in the basolateral amygdala and CA1 region: Possible role of

calcium channel subunits. J Neurophysiol 99:958–968.15. Johnson LR, Farb C, Morrison JH,McEwen BS, LeDoux JE (2005) Localization of glucocorticoid

receptors at postsynaptic membranes in the lateral amygdala. Neuroscience 136:289–299.16. Erdmann G, Schütz G, Berger S (2007) Inducible gene inactivation in neurons of the

adult mouse forebrain. BMC Neurosci, 10.1186/1471-2202-8-63.17. Di S, Malcher-Lopes R, Halmos KC, Tasker JG (2003) Nongenomic glucocorticoid

inhibition via endocannabinoid release in the hypothalamus: A fast feedback

mechanism. J Neurosci 23:4850–4857.18. Campolongo P, et al. (2009) Endocannabinoids in the rat basolateral amygdala

enhance memory consolidation and enable glucocorticoid modulation of memory.

Proc Natl Acad Sci USA 106:4888–4893.19. Vasudevan N, Kow LM, Pfaff DW (2001) Early membrane estrogenic effects required

for full expression of slower genomic actions in a nerve cell line. Proc Natl Acad Sci

USA 98:12267–12271.20. Buchanan TW, Lovallo WR (2001) Enhanced memory for emotional material following

stress-level cortisol treatment in humans. Psychoneuroendocrinology 26:307–317.21. Abraham WC, Bear MF (1996) Metaplasticity: The plasticity of synaptic plasticity.

Trends Neurosci 19:126–130.22. Yehuda R (2006) Advances in understanding neuroendocrine alterations in PTSD and

their therapeutic implications. Ann NY Acad Sci 1071:137–166.23. Berger S, et al. (2006) Loss of the limbic mineralocorticoid receptor impairs behavioral

plasticity. Proc Natl Acad Sci USA 103:195–200.24. Tronche F, et al. (1999) Disruption of the glucocorticoid receptor gene in the nervous

system results in reduced anxiety. Nat Genet 23:99–103.

14454 | www.pnas.org/cgi/doi/10.1073/pnas.0914381107 Karst et al.