Mineralocorticoid receptors are indispensable for nongenomic modulation of hippocampal glutamate transmission by corticosterone

Henk Karst*†, Stefan Berger‡, Marc Turiault§, Francois Tronche§,Gu¨ nther Schu¨ tz‡, and Marian Joe¨ ls*

*Swammerdam Institute for Life Sciences, Center for Neurosciences (SILS–CNS), University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, The Netherlands; ‡German Cancer Research Institute, 69120 Heidelberg, Germany; and §Centre National de la Recherche Scientifique FRE 2401, College de France, 75231 Paris Cedex, France

Edited by Bruce S. McEwen, The Rockefeller University, New York, NY, and approved October 26, 2005 (received for review August 31, 2005) The adrenal hormone corticosterone transcriptionally regulates housed in cages with a light͞dark cycle of 12 h (lights on at 0800 responsive genes in the rodent hippocampus through nuclear hours). Food and water were given ad libitum. The experiments mineralocorticoid and glucocorticoid receptors. Via this genomic were carried out with permission of the local Animal Committee pathway the hormone alters properties of hippocampal cells (file no. DED 91). One mouse per day was decapitated under rest slowly and for a prolonged period. Here we report that cortico- around 0930 hours, i.e., when plasma corticosterone levels are sterone also rapidly and reversibly changes hippocampal signaling. low (ref. 5; Ϸ2 ␮g͞dl). Stress levels of the hormone enhance the frequency of miniature A limited number of experiments was performed in brain- excitatory postsynaptic potentials in CA1 pyramidal neurons and specific GR (12) and MR knockout mice. Details about the reduce paired-pulse facilitation, pointing to a hormone-dependent generation of MR mutants are described in Supporting Text, enhancement of glutamate-release probability. The rapid effect by which is published as supporting information on the PNAS web corticosterone is accomplished through a nongenomic pathway site. In these two mutant lines, immunoreactivity of the GR and involving membrane-located receptors. Unexpectedly, the rapid MR protein, respectively, was lost in all CA1 neurons, which did effect critically depends on the classical mineralocorticoid receptor, express the proteins in control animals. In view of the elevated as evidenced by the effectiveness of agonists, antagonists, and corticosterone levels in GR mutants, both the mutant mice and brain-specific inactivation of the mineralocorticoid but not the their control littermates (GRloxP/loxP) were adrenalectomized, glucocorticoid receptor gene. Rapid actions by corticosterone which in mice resulted in low (Ϸ1 ␮g͞dl) levels of circulating would allow the brain to change its function within minutes after corticosterone, not unlike the levels in adrenally intact mice, stress-induced elevations of corticosteroid levels, in addition to which were decapitated in the morning under rest. responding later through gene-mediated signaling pathways. Slice Preparation and Recording. On the day of the experiment, the ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor ͉ CA1 animal was decapitated, and the brain was removed from the hippocampus ͉ glucocorticoid receptor knockout ͉ miniature excitatory skull and stored in continuously gassed medium. Transverse postsynaptic current ͉ mineralocorticoid receptor knockout slices of the hippocampus were made with a tissue chopper and stored at room temperature in medium containing 124 mM he release of corticosterone, i.e., the prevailing endogenous NaCl, 3.5 mM KCl, 1.25 mM NaH2PO4, 1.5 mM MgSO4,2mM ϭ Tadrenal corticosteroid in rats and mice, is markedly en- CaCl, 25 mM NaHCO3, and 10 mM glucose (osmolarity 300 hanced after exposure to a stressor. Hormone levels rise within mOsm; pH 7.4). One slice at a time was fully submerged in a minutes and return to baseline Ϸ60–90 min after the stress (1). recording chamber and continuously perfused with the medium Corticosterone can quickly enter the brain and bind to two (32°C, 2–3 ml͞s), as described in ref. 13. Bicuculline methiodide subtypes of discretely localized receptors, the high-affinity min- (20 ␮M, Sigma) was added to block GABAA receptor-mediated eralocorticoid receptor (MR) and the lower-affinity glucocor- signals. ticoid receptor (GR) (2). Hippocampal CA1 neurons abundantly Whole-cell patch clamp recordings were made with an Axo- express both receptor types. Activated corticosteroid receptors patch 200B amplifier (Axon Instruments, Union City, CA) by are known to modify transcription of responsive genes, either using borosilicate glass electrodes (1.5-mm outer diameter; through DNA binding of homodimers or through protein– Hilgenberg, Malsfeld, Germany; pulled on a Sutter micropipette protein interactions with other transcription factors (3, 4). In this puller), with an impedance of 3–4 M⍀. The intracellular pipette way, hippocampal cell properties, like calcium influx, were found solution contained 120 mM Cs methane sulfonate, 17.5 mM to be altered in a slow but persistent manner (5). In recent years, CsCl, 10 mM Hepes, 2 mM MgATP, 0.1 mM NaGTP, 5 mM though, preliminary evidence has accumulated that corticoste- BAPTA, and 10 mM QX-314 (osmolarity ϭ 295 mOsm; pH 7.4), rone can also induce rapid effects in brain (6–10), which would adjusted with CsOH. BAPTA was obtained from Molecular vastly expand the time window over which adrenal hormones can Probes, the sodium channel blocker QX-314 was from Alomone affect brain function. Although in amphibian brain a specific (Jerusalem), and all other chemicals were from Sigma. Under membrane receptor was isolated mediating rapid behavioral visual control the recording electrode was directed toward a CA1 effects (11), the underlying mechanism and presumed mem- neuron by using positive pressure. Once a patch electrode was brane receptor involved in rapid effects of the mammalian sealed on the cell (Ϸ1G⍀) the membrane patch under the hormone remained unresolved. We here demonstrate that a rapid nongenomic effect by corticosterone through membrane- located receptors critically depends on the MR, which hitherto Conflict of interest statement: No conflict declared. was regarded only as a nuclear receptor. This paper was submitted directly (Track II) to the PNAS office. Abbreviations: EPSC, excitatory postsynaptic current; mEPSC, miniature EPSC; MR, miner- Methods alocorticoid receptor; GR, glucocorticoid receptor. Animals. All experiments, unless stated otherwise, were per- †To whom correspondence should be addressed. E-mail: [email protected]. formed on male C57BL͞6 mice, Ϸ6 weeks of age and group- © 2005 by The National Academy of Sciences of the USA

19204–19207 ͉ PNAS ͉ December 27, 2005 ͉ vol. 102 ͉ no. 52 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0507572102 Downloaded by guest on September 23, 2021 Fig. 1. Corticosterone rapidly and reversibly enhances mEPSC frequency in hippocampal cells. (A) Typical example showing enhanced mEPSC frequency in a CA1 pyramidal cell during 100 nM corticosterone (cort) application (Middle) compared with the control situation (Top). Bottom shows the typical shape of an mEPSC. (B) Example showing that mEPSC frequency is rapidly and reversibly enhanced by corticosterone but not by vehicle (0.009% ethanol). (C) Cumulative frequency histogram of the mEPSC frequency in a CA1 hippocampal cell, showing that the mEPSC frequency is enhanced in the presence of corticosterone and returns to pretreatment levels after corticosterone has been washed out. (D) Percentual increase in mEPSC frequency caused by various concentrations of corticosterone (filled circles, based on n ϭ 3–5 cells for each concentration), showing that 10 nM corticosterone, but not lower concentrations of corticosterone, induces a significant (P Ͻ 0.05) increase in mEPSC frequency. The membrane-impermeable BSA–corticosterone conjugate (100 nM; n ϭ 5) also resulted in a marked increase in mEPSC frequency. In the presence of the protein synthesis inhibitor cycloheximide (100 nM; n ϭ 5), corticosterone was still able to enhance the mEPSC frequency. Symbols represent the mean ϩ SEM values.

electrode was ruptured and the cell was held at a holding receptor blocker CNQX or the selective ␣-amino-3-hydroxy-5- potential of Ϫ70 mV. Recordings with an uncompensated series methyl-4-isoxazolepropionic acid (AMPA) receptor blocker resistance of Ͻ2.5 times the pipette resistance were accepted for GYKI 53655 was perfused to confirm that the mEPSCs were analysis. Data acquisition was performed with PCLAMP 8.2 and indeed mediated by AMPA receptors. The digitized data (stored analyzed with CLAMPFIT 8.2 and STRATHCLYDE software (J. on PC via Digidata interface) were analyzed off-line by using Dempster, University of Strathclyde, Glasgow, U.K.). STRATHCLYDE software with detection threshold levels set above 5 pA. The currents were identified as mEPSCs when the rise time Stimulus Evoked of Excitatory Postsynaptic Currents (EPSCs). A bi- was faster than the decay time (13). Of all cells measured, the polar stainless steel stimulus electrode was placed in the Schaffer following mEPSC characteristics were determined: inter- collaterals. Biphasic stimuli were applied through a stimulus mEPSC interval and frequency, rise time, peak amplitude, and isolator driven by PCLAMP 8.2. Input–output curves of EPSCs ␶ of decay. The decay of each mEPSC was fitted with a evoked in CA1 neurons were made at holding potential (Ϫ70 monoexponential curve using the WHOLECELL program of the mV) by stimuli of increasing intensities (from 16 ␮A to 500 ␮A), STRATHCLYDE software. This program uses the Levenberg– given once every 10 seconds. Evoked EPSCs were recorded with Marquardt algorithm to iteratively minimize the sum of the a sampling frequency of 10 kHz. Signals were stored and squared differences between the theoretical curve and the data corrected off-line for leak. Input–output curves were fitted with curve. As criterion for the goodness of the fit the residual a Boltzmann equation: R(i) ϭ Rmax͞[1 ϩ exp{(i Ϫ iH)͞IC}], standard deviation should be Ͻ0.3. Fitting with a biexponential where Rmax is the maximal evoked current, iH is the half maximal curve instead of a monoexponential curve did not increase stimulus intensity, and IC is proportional to the slope. Based on goodness of the fit (data not shown). this curve the half maximal stimulus intensity was determined. NEUROSCIENCE This intensity was used to record responses to paired pulse Statistics. Rapid effects on frequency, amplitude, and decay time stimulation (interstimulus interval, 100 ms). were analyzed with a two-tailed paired Student t test. In all cases, significance was set at P Ͻ 0.05. Miniature EPSCs (mEPSCs). mEPSCs were always recorded in the presence of 0.5 ␮M TTX, either added to the buffer after Results recording of evoked EPSCs in the same cell or present during the mEPSCs were recorded from 82 CA1 pyramidal cells in acutely entire experiment (i.e., in case evoked EPSCs were not re- prepared adult mouse hippocampal slices (Fig. 1A). These corded). At a holding potential of Ϫ70 mV, mEPSCs were currents reflect the spontaneous fusion of glutamate-containing recorded for 5 min. During some recordings the non-NMDA vesicles with the presynaptic membrane (14). All currents were

Karst et al. PNAS ͉ December 27, 2005 ͉ vol. 102 ͉ no. 52 ͉ 19205 Downloaded by guest on September 23, 2021 Table 1. Effect of corticosterone on frequency, amplitude, and decay time of mEPSCs in CA1 hippocampal neurons Condition mEPSC frequency, Hz mEPSC amplitude, pA ␶ of decay, s

Before corticosterone 0.85 Ϯ 0.14 22.8 Ϯ 2.3 17.5 Ϯ 1.6 During corticosterone 1.33 Ϯ 0.25* 24.5 Ϯ 1.9 15.8 Ϯ 0.4

All values represent the mean Ϯ SEM, based on a total of 3,242 events from five cells. Corticosterone significantly (*, P Ͻ 0.05) enhanced the mEPSC frequency (determined between 5 and 10 min after application was started), whereas the amplitude and time constant of the decay (based on a monoexponential fit) remained unaffected. Control data were collected between 5 and 0 min before corticosterone application was started.

recorded at a holding potential of Ϫ70 mV, when NMDA effect of corticosterone, because administration of a cortico- receptors are presumably blocked by Mg2ϩ ions (15). The sterone–BSA conjugate (100 nM), which cannot pass the mem- exclusive involvement of ␣-amino-3-hydroxy-5-methyl-4- brane, increased the mEPSC frequency (Fig. 1D; P ϭ 0.03) with isoxazolepropionic acid (AMPA) receptors was further evi- an efficacy that was comparable to that of corticosterone itself. denced by effective reversible blockade of the mEPSCs by the Increases in mEPSC frequency, but not amplitude or kinetics, non-NMDA receptor antagonist CNQX and the selective point to a change in presynaptic properties of glutamate trans- AMPA receptor antagonist GYKI 53655 (data not shown, see mission (19). To distinguish between an increased release prob- refs. 13, 16, and 17). ability and an enhanced number of synaptic contacts we exam- Bath application of 100 nM corticosterone rapidly and revers- ined paired-pulse responses to stimulation of the Schaffer ibly enhanced the frequency of mEPSCs (see examples in Fig. 1 collateral afferents (interstimulus interval, 100 ms). We ob- A–C). On average, corticosterone induced a 60% increase in the served that corticosterone decreases the ratio of the second to mEPSC frequency (Table 1). In the hippocampus, enhancement the first response (before, 1.20 Ϯ 0.03; during, 1.00 Ϯ 0.04, n ϭ of mEPSC frequency was seen already at 10 nM corticosterone 4; P ϭ 0.04). This finding is compatible with the notion that the (Fig. 1D). The mEPSC amplitude was not affected by the hormone increases the release probability for glutamate (20). In hormone (Table 1). Similarly, kinetic properties like the time agreement, a recent study using microdialysis demonstrated a constant of the decay were unaffected (Table 1). The enhance- rapid, nongenomic increase of glutamate but not GABA release ment of glutamate transmission may be hippocampus-specific, by a high dose of corticosterone (8). Such a rapid increase in because 100 nM corticosterone rapidly reduced mEPSC fre- glutamate release potentially enhances excitability in the CA1 quency in parvocellular neurons of the hypothalamic paraven- region, but it should be emphasized that overall excitability tricular nucleus (38.9 Ϯ 15.9%, n ϭ 3), in line with an earlier depends not only on processes taking place in presynaptic report (9). terminals as presently studied, but also on other factors, e.g., the The rapid onset and reversibility of the effect indicate that a stability of synaptic contacts that are located on higher-order nongenomic pathway underlies the hormone action. In agree- dendritic branches and intrinsic network properties related to ment, the increase in mEPSC frequency by corticosterone was of feedforward or feedback inhibition. comparable magnitude when tested in the presence of a trans- To determine whether ‘‘classical’’ steroid receptors are in- lation inhibitor, cycloheximide (Fig. 1D; P ϭ 0.04). Earlier volved in these rapid, nongenomic effects of corticosterone, we studies in nonneuronal tissues have shown that nongenomic used selective receptor agonists and antagonists as well as steroid effects can be mediated by a receptor located in the brain-specific GR or MR knockout mice. Based on the relatively membrane (18). This also appears to be the case for the present high threshold concentration, we assumed that the rapid effects

Fig. 2. Increased mEPSC frequency during corticosterone application is mediated by the MR and not the GR. (A) The selective GR agonist RU 28362 (100 nM) does not increase the mEPSC frequency (n ϭ 4). The GR antagonist RU 38486 (500 nM; n ϭ 4) does not significantly affect the percentual increase in mEPSC frequency caused by corticosterone when compared with the response evoked by corticosterone alone (shown on the far left). By contrast, the endogenous mineralocorticoid aldosterone (10 nM; n ϭ 5), applied in the presence of the GR antagonist RU 38486, potently increased the mEPSC frequency. Additional application of the MR antagonist spironolactone (100 nM; n ϭ 5) completely abolished the response to aldosterone. (B) Corticosterone (100 nM) significantly enhances the mEPSC frequency in CA1 pyramidal cells of brain-specific GR knockout mice (GRNesCre)(n ϭ 4 cells) as well as of controls (GRloxP/loxP)(n ϭ 4). In cells of brain-specific MR knockout mice (MRCamKIICre)(n ϭ 6), however, corticosterone application was ineffective, and normal effects were seen in control mice (MRloxP/loxP)(n ϭ 6). All data represent the percentual increase in mEPSC frequency, expressed as the ratio of the averaged frequency determined between 5 and 10 min after administration of the agonist and the averaged frequency between 5 and 0 min before administration was started (*, P Ͻ 0.05). Details on the transgenesis are provided in Supporting Text.

19206 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0507572102 Karst et al. Downloaded by guest on September 23, 2021 were mediated by GR rather than MR. However, the highly Because corticosterone does not have to pass the membrane to selective GR agonist RU 28362 (21) did not increase mEPSC accomplish these effects in hippocampus, it is likely that the frequency (Fig. 2A). Also, the effects by corticosterone were not receptor is localized within the membrane. Imaging and immuno- significantly blocked by RU 38486, which interferes with trans- cytochemical studies so far have demonstrated the MR to be activation via the GR (21) (Fig. 2A). Finally, in hippocampal present primarily in the nuclear compartment (24–26), although slices prepared from brain-specific GR mutant mice (12), the membrane localization was reported with immunoelectron micros- mEPSC frequency was still significantly enhanced (Fig. 2B). copy for human kidney cells (27). We propose that part of the brain Evidently, the GR is not involved in these rapid hormone effects. MRs (the degree of which may depend on long-term averages of By contrast, aldosterone, which in rodents has a higher affinity circulating hormone levels) can shuttle from the nucleus͞cytoplasm for MR than GR (21), markedly enhanced mEPSC frequency, at to the cell membrane. Shuttling of steroid receptors is not unprec- a concentration (10 nM) that induced only threshold effects with edented, because incorporation of a small fraction of the estrogen corticosterone (Fig. 2A). In view of this higher sensitivity to receptors (Ͻ10%) into the cell membrane has been demonstrated aldosterone than corticosterone and the fact that aldosterone (28). This mechanism could explain the rapid effects on excitability was tested in the presence of the GR antagonist RU 38486, of hippocampal (29) and hypothalamic neurons (30) seen with involvement of an MR rather than GR is indicated. In agree- estrogens. Shuttling of the receptor may involve rearrangement of ment, the effect of aldosterone (Fig. 2A) in hippocampus was chaperone proteins, such as heat-shock protein 90, and 3D adap- completely blocked by spironolactone, an MR antagonist (22). tation of the receptor. These as well as other factors involving the Most importantly, no effect at all of corticosterone on mEPSC receptor structure could explain the apparent decrease in affinity of frequency was observed in CA1 cells of forebrain-specific MR the receptor (31). knockout mice as opposed to the controls (Fig. 2B). The requirement of at least 10 nM corticosterone to induce Discussion rapid effects on hippocampal glutamate transmission indicates that such effects will not occur with basal levels of the hormone The present study demonstrates a fast-onset, rapidly reversible, Ϸ and nongenomic enhancement by corticosterone of glutamate but only when hormone levels are elevated, i.e., 10–60 min transmission in the CA1 hippocampal area. With the use of a after stress exposure or possibly during the circadian peak (32). unique forebrain-specific MR knockout mouse model we dem- The MR could thus serve as a ‘‘cortico-sensor’’ in the brain, onstrated that these nongenomic effects critically depend on a rapidly changing excitatory transmission while corticosteroid gene that encodes the MR. Until now this receptor was thought levels are enhanced. This mechanism would supply the brain to mediate only slow actions in brain, through transcriptional with the means to react quickly to stress through the MR, next regulation of responsive genes. The threshold corticosterone to other rapid stress-activated systems involving noradrenaline concentration for rapid effects was presently found to be Ϸ10 and corticotropin-releasing hormone. Gene-mediated stress ef- nM, whereas earlier electrophysiological investigations showed fects by the hormone (13), which in hippocampus typically that 10- to 20-fold lower concentrations suffice to activate the develop with a delay of 1–2 h and mostly through the GR (23), intracellular MR (23). This lower apparent affinity of the MRs would next adapt hippocampal activity when circulating hor- involved in rapid corticosteroid effects could lend a new meaning mone levels have normalized again after stress and, thus, when to this receptor, of which the role in the stress response has rapid MR effects have subsided. Through this binary mode of remained somewhat difficult to understand given its very high action, one hormone (corticosterone) could alter hippocampal affinity and hence considerable occupancy already with low activity over a very prolonged period, during as well as after circulating levels of the hormone. exposure to a stressful situation.

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