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. 2011 Dec 23;590(Pt 4):875–886. doi: 10.1113/jphysiol.2011.223784

Acute stress induces down-regulation of large-conductance Ca2+-activated potassium channels in the lateral amygdala

Yan-yan Guo 1, Shui-bing Liu 1, Guang-Bin Cui 2, Lan Ma 1, Bin Feng 1, Jiang-hao Xing 1, Qi Yang 1, Xiao-qiang Li 1, Yu-mei Wu 1, Li-ze Xiong 3, Weiqi Zhang 4, Ming-gao Zhao 1
PMCID: PMC3381316  PMID: 22199169

Abstract

Large-conductance Ca2+-activated potassium channels (BKCa) are highly expressed in the lateral amygdala (LA), which is closely involved in assigning stress disorders, but data on their role in the neuronal circuits of stress disorders are limited. In the present study, a significant reduction in BKCa channel expression in the amygdala of mice accompanied anxiety-like behaviour induced by acute stress. Whole-cell patch-clamp recordings from LA neurons of the anxious animals revealed a pronounced reduction in the fast after-hyperpolarization (fAHP) of action potentials mediated by BKCa channels that led to hyperexcitability of the LA neurons. Activation of BKCa channels in the LA reversed stress-induced anxiety-like behaviour after stress. Furthermore, down-regulated BKCa channels notably increased the evoked NMDA receptor-mediated excitatory postsynaptic potentials at the thalamo-LA synapses. These data demonstrate, for the first time, that restraint stress-induced anxiety-like behaviour could at least partly be explained by alterations in the functional BKCa channels in the LA.

Key points

  • Stress can lead to the development of behavioural disorders associated with cognitive impairments, depression and anxiety.

  • Large-conductance Ca2+-activated potassium channels (BKCa) are highly expressed in the brain. Here we found that acute stress induced a significant reduction in BKCa channel expression in the amygdala of mice, which accompanied anxiety-like behaviours.

  • Activation of BKCa channels in the amygdala could reverse the stress-induced anxiety-like behaviours. This research may help us understand the underlying mechanisms of anxiety-like behaviour induced by acute stress.

Introduction

Stress initiates a series of neuronal responses and can lead to the development of behavioural disorders associated with cognitive impairments, depression and anxiety (Shekhar et al. 2005; Roozendaal et al. 2009). Growing evidence has implicated the amygdala in mediation of emotional disorders (Pawlak et al. 2003; Ehrlich et al. 2009; Ressler, 2010). Chronic stress causes amygdala hyperexcitability and the effects of stress on the excitability are occluded by agents that block calcium-activated potassium channels (KCa) and reversed by pharmacological enhancement of KCa (Rosenkranz et al. 2010). Large-conductance Ca2+-activated potassium channels (BKCa) are widely expressed in neurons of the brain (Sah, 1996; Poolos & Johnston, 1999; Martinez-Pinna et al. 2000), including those in the amygdala (Faber & Sah, 2003). BKCa channel activation is critical for shaping action potentials (APs) by substantially contributing to their repolarization and fast after-hyperpolarization (fAHP) (Lancaster et al. 1991; Faber & Sah, 2003; Haghdoost-Yazdi et al. 2008; Womack et al. 2009). The fAHP contributes most strongly to repolarization of the APs at the beginning of a train, as the channel is inactivated during subsequent APs (Shao et al. 1999). Overall, the BKCa channels can regulate firing rates and play a crucial role in the neuronal circuitry in many types of neurons (Smith et al. 2002; Faber & Sah, 2003; Womack et al. 2009). The functional role of the BKCa current has been demonstrated in epilepsy (Du et al. 2005), the vestibular-ocular reflex (Nelson et al. 2003), circadian behavioural rhythms (Meredith et al. 2006), extinction of fear conditioning (Santini et al. 2008) and hippocampus-dependent learning (Matthews et al. 2008). Whether or not BKCa channels play a role in stress-induced anxiety, however, is unclear.

The NMDA receptor (NMDAR) is an important mediator of synaptic plasticity and plays a central role in the neurobiological mechanisms of emotionality including fear, anxiety and depression (Bliss & Collingridge, 1993; Maren, 1999; Zhao et al. 2005). BKCa channels represent a potential target for NMDAR-mediated Ca2+ influx and functionally couple to NMDARs in granule cells in the olfactory bulb to evoke a slow inhibitory postsynaptic current (Isaacson & Murphy, 2001). However, functional coupling properties of BKCa channels and NMDARs in the lateral amygdala (LA) and the changes in the BKCa channels of neuronal circuits in stress disorders are limited. The present study found that BKCa channels promoted stress-induced neuronal remodelling in the LA, and that their down-regulation was critical to the development of anxiety after stress.

Methods

Animals and restraint stress experiments

Six- to eight-week-old C57BL/6 male mice were used. All animals were housed under a 12 h dark–12 h light cycle with food and water provided ad libitum. Acute stress was induced by restraining the mice in well-ventilated Perspex restraining tubes for 2 h, during which the animals were not physically compressed or experience pain (Chotiwat & Harris, 2006). The restrained mice were divided into two subgroups: Group 1 was exposed to a single 2 h restraint (Restraint 1), whereas Group 2 was exposed to two 2 h of restraints on each of two consecutive days (Restraint 2). After restraints, the mice were returned to their home cages and given food and water ad libitum. Non-restrained controls were placed in a Perspex cage in the same experimental room during the restraining period. One day later, the mice were used for behavioural tests, brain slice recording and Western blot analysis. All behavioural testing occurred between 09.00 and 12.00 h on the designated day of experiment. The Fourth Military Medical University Animal Care and Use Committee approved the animal protocols. All animal experiments conform to the principles of UK regulations. The authors have read, and the experiments comply with the policies and regulations of The Journal of Physiology given by Drummond (2009).

Elevated plus maze

The elevated plus maze (EPM) was conducted as described in Liu et al. (2007). The apparatus comprised two open arms (25 cm × 8 cm × 0.5 cm) and two closed arms (25 cm × 8 cm × 12 cm) that extended from a common central platform (8 cm × 8 cm). The apparatus was elevated to a height of 50 cm above the floor. Mice were allowed to habituate to the testing room for 2 days before the test, and were pre-treated with gentle handling two times per day to eliminate their nervousness. For each test, individual animals were placed in the centre square, facing an open arm, and allowed to move freely for 5 min. Mice were videotaped using a camera fixed above the maze and analysed with a video-tracking system. Open and closed arm entries (all four paws in an arm) were scored by an experienced observer. The number of entries and time spent in each arm were recorded.

Open-field test

The open-field (OP) test was conducted as described in Liu et al. (2007). The open field was a square arena (30 cm × 30 cm × 30 cm) with clear Plexiglas walls and floor, and was placed inside an isolation chamber with dim illumination and a fan. Mice were placed in the centre of the box and allowed to freely explore for a 10 min period. Mice were videotaped using a camera fixed above the floor and analysed with a video-tracking system. The ‘centre’ field is defined as the central 15 cm × 15 cm area of the open field, one-fourth of the total area. Each mouse was placed in the centre of the open field, and its activity was measured for 30 min.

Amygdala cannulation and microinjection

Mice were anaesthetized with an i.p. injection of 3 ml kg−1 of a mixture of ketamine (30 mg ml−1) and xylazine (3 mg ml−1). Depth of anaesthesia was assessed by monitoring breathing rate and determining reflex responses following pinching of the foot. A midline incision was made to expose the top of the skull. A dental drill was used to make a small hole (approx. 2 mm diameter). A 24-gauge guide cannula was bilaterally implanted into the LA (–1.4 mm anterior to bregma, 3.3 mm lateral from the midline, and 4.4 mm beneath the surface of the skull). The mice were given at least 2 weeks to recover after cannula implantation. The 30-gauge injection cannula used was 0.1 mm lower than the guide cannula. For intra-amygdala infusion, the animals were placed individually in an induction chamber, and anaesthesia was induced with 2.5% isoflurane (JiuPai, Shijiazhuang, China) in 100% oxygen with a delivery rate of 0.5 l min−1 until loss of righting reflex. Anaesthesia was then maintained with 1.5% isoflurane in 100% oxygen with a flow of 0.5 l min−1 delivered by face mask. The selective BKCa channel agonist NS1619 (10 μm, 0.5 μl) (Sigma, St Louis, MO, USA) or saline vehicle (0.5 μl) was bilaterally delivered at 0.5 μl min−1 using a syringe driven by an infusion pump (Harvard Apparatus, Inc., South Natick, MA, USA). After infusion, the cannula was left in place for an additional 2 min to allow the solution to diffuse away from the cannula tip. Thirty minutes later, the mice were subjected to elevated plus maze (EPM) and open-field tests. Then the animals were killed by being rendered unconscious by 4% isoflurane in air and then killed by cervical dislocation. To confirm the injection site of LA, the whole brains were fixed with 4% paraformaldehyde and dehydrated through an ascending alcohol series. The brain tissues were sliced on a freezing microtome (Leica, Nussloch, Germany), and 30 μm coronal sections containing the amygdala were collected. The sections were mounted on glass slides and stained with haematoxylin and eosin. Images were taken using an Olympus light microscope equipped with a CCD camera (Olympus, Japan). The Fourth Military Medical University Animal Care and Use Committee approved the animal protocols.

Western blot analysis

Western blot analysis was performed as described previously (Chen et al. 2008). Tissue samples from the bilateral LA were dissected from the brain slices under the anatomical microscope. Equal amounts of protein (50 μg) were separated and electrotransferred onto PDVF membranes (Invitrogen), which were probed with antibody for BKCa (dilution ratio 1:400, Alomone Labs, Jerusalem, Israel; product no., APC-021) and with β-actin (dilution ratio 1:10000, Sigma) as loading control. For data quantitation, band intensity was expressed relative to the loading control (β-actin).The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG for the primary antibodies), and bands were visualized using an ECL system (Perkin Elmer).

Whole-cell patch-clamp recording

The animals were killed by being rendered unconscious by 4% isoflurane in air and then killed by cervical dislocation. The brains were rapidly removed into ice-cold artificial cerebrospinal fluid (ACSF) containing 124 mm NaCl, 25 mm NaHCO3, 2.5 mm KCl, 1 mm KH2PO4, 2 mm CaCl2, 2 mm MgSO4 and 10 mm glucose. Transverse slices (300 μm) containing the LA were cut and transferred to a room temperature-submerged recovery chamber with oxygenated (95% O2 and 5% CO2) ACSF. After 1 h of recovery, slices were placed in a recording chamber on the stage of an Olympus microscope with infrared digital interference contrast optics for visualization of whole-cell patch-clamp recordings. EPSCs were recorded from cells in the LA with an Axon 200B amplifier (Axon Instruments, Union City, CA, USA). Electrical stimulations (0.2 ms duration) were delivered by a bipolar tungsten stimulating electrode placed in the internal capsule (thalamic inputs) (Tsvetkov et al. 2004). For firing rate recording, recording pipettes (3–5 MΩ) were filled with solution containing 145 mm potassium gluconate, 5 mm NaCl, 1 mm MgCl2, 0.2 mm EGTA, 10 mm Hepes, 2 mm Mg-ATP and 0.1 mm Na3-GTP, adjusted to pH 7.2 with KOH (280–300 mosmol l−1). Interneurons and pyramidal neurons were identified by their different firing patterns and morphology. The typical firing pattern of pyramidal neurons showed significant firing frequency adaptation (Tsvetkov et al. 2002), whereas interneurons showed fast-spiking APs followed by pronounced hyperpolarization, lower rheobase current and higher input resistance (Wu et al. 2007). In the present study, pyramidal neurons in the LA were recorded. Action potentials were elicited in the presence of 50 μm CNQX and 100 μm picrotoxin by positive current injection (100–300 pA) in current-clamp mode. fAHP was defined as the difference between spike threshold and the most negative potential after each AP in a train. AP duration was measured as the spike width at half-maximal amplitude. NMDAR-mediated components of excitatory postsynaptic potentials (EPSPs) elicited by three pulses at 100 Hz were pharmacologically isolated in magnesium-free ACSF containing 1 μm glycine, 20 μm CNQX and 100 μm picrotoxin. Access resistance (15–30 MΩ) was monitored throughout the experiment. Data were discarded if access resistance changed >15% during an experiment and the resting membrane potential was more depolarized than –60 mV.

Data analysis

Results were expressed as the mean ± SEM. Statistical comparisons were performed using one-way ANOVA and the Student–Newman–Keuls test for post hoc comparisons. In all cases, P < 0.05 was considered statistically significant.

Results

Enhanced anxiety by acute restraint stress

Acute restraint stress decreased the time spent in open arms and open arm entries of the EPM compared with the naïve control animals (Fig. 1A and B). Time spent in the central areas in the OP tests also significantly decreased (Fig. 1C and D). These changes were even more evident in the mice subjected to Restraint 2. However, neither the total number of arm entries in the EPM tests nor the total distance travelled in the OP tests changed compared with the naïve controls (Fig. 1B and D), showing that there was no difference in locomotor activity by acute stress between the groups. These data demonstrate that exposure to acute stress enhances anxiety-like behaviour in mice.

Figure 1. Enhanced anxiety by acute restraint stress.

Figure 1

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A, representative traces showing the movement of control mice and mice subjected to single and two restraints in the EPM for 5 min. B, mice subjected to single (n = 6) and two restraints (n = 6) spent less time in the open arms of EPM and showed a decrease in open arm entries compared with naïve control mice (n = 8). Total number of arm entries in the EPM tests was not changed compared with control mice. C, representative traces showing the movement of control mice and mice subjected to single and two restraints in the open-field test for 30 min. D, mice subjected to single or two restraints spent significantly less time in the central areas in the OP test. Total distance travelled in the OP tests was not changed compared with the control animals. *P < 0.05, **P < 0.01 versus the control.

BKCa channels modulate the excitability of pyramidal neurons in the LA

It has been previously reported that blocking the BKCa channel increases firing output to a given input current (Nelson et al. 2003) and the BKCa channel contributes to the repolarization of the action potential (Faber & Sah, 2003). To test the direct contribution of BKCa channels to excitability of the pyramidal neurons, action potentials (APs) were elicited in the presence of CNQX (50 μm) and picrotoxin (100 μm) by positive current injection (100–200 pA) to generate six APs in 500 ms in the current-clamp recording (Fig. 2A). Comparisons of the firing properties (before the drug perfusion) were always made between equivalent firing frequency (i.e. 6 APs in a burst) to avoid the original variation of AP duration. The fAHP contributes most strongly to repolarization of the APs at the beginning of a train, as the channel is inactivated during subsequent APs (Shao et al. 1999; Matthews et al. 2008). Only the first three APs were analysed in this study. Blocking the BKCa channel with iberotoxin (IBTX, 100 nm) resulted in a notable reduction in the negative peak of the fAHP, an increase in AP half-width, and a prolonged decay time for the first two APs (Fig. 2B–D). The results implicate the functional roles of BKCa channel in the control of intrinsic excitability in the pyramidal neurons. Furthermore, the activation of BKCa channels depended on cytoplasmic Ca2+ elevation. The Ca2+ chelator BAPTA (10 mm) via the patch-clamp pipette abolished the alteration by BKCa channel in the fAHP (Fig. 2E), AP duration (Fig. 2F) and AP decay time (Fig. 2G).

Figure 2. Role of BKCa channels in the firing properties.

Figure 2

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A, left: representative traces showing the firing property in the neurons from LA of a naïve mouse response to a 135 pA depolarizing current injection (500 ms). Right: representative recording of the first spike in the neurons from control, iberotoxin- and NS1619-treated slices. fAHP was defined as the difference between spike threshold and the most negative potential after each AP in a train. AP duration was measured as the spike width at half-maximal amplitude. First action potentials have been superimposed to show the extent of spike broadening and fAHP. B, iberotoxin (n = 12) resulted in a notable reduction in the negative-going peak of the fAHP, while NS1619 (n = 14) caused a notable increase in fAHP of the first 3 spikes in a train compared with the saline control (n = 15). C, iberotoxin (n = 12) increased AP half-width of the first two APs, while NS1619 (n = 14) decreased AP half-width compared with the saline control (n = 15). D, iberotoxin (n = 12) increased AP decay time of the first two APs, while NS1619 (n = 14) decreased AP half-width compared with the saline control (n = 15). E–G, changes of peak fAHP (E), half-width (F) and decay time (G) of the first 3 spikes by IBTX (n = 8) or NS1619 (n = 9) were occluded in the presence of BAPTA (10 mm) loaded in internal solution. *P < 0.05 versus the control.

Down-regulation of BKCa channels in the amygdala after acute stress

As the LA is an essential component of the circuitry underlying anxiety (Davis, 1992; Gross & Hen, 2004; Ehrlich et al. 2009) and learning modulates BKCa channel-mediated fAHP (Matthews et al. 2008), BKCa channels were hypothesized to be involved in stress-induced anxiety. We found that there was a little depolarization of the resting membrane potential (RMP) in the pyramidal neurons from the restrained mice; however, the RMP was not significantly different between the restrained mice and naïve control mice (Fig. 3A). Then, we tested the effects of acute stress on the neuronal excitability in the LA. In mice that were exposed to acute stress, LA pyramidal neurons displayed a greater basal firing rate than in control rats. The firing frequency to a given input current (100 pA) increased significantly in the restrained mice (Fig. 3A and B). By using an occlusion approach, the stress-related difference in firing frequency to a given step input was shown to depend on BKCa channel activity by bath-applied IBTX (100 nm). First, equivalent firing frequency (i.e. 7 APs in a burst) was elicited by varied current injection (110–140 pA) to avoid possible influences by the firing frequency. We found that the increase in firing frequency by IBTX was greater in cells from the naïve animals than in those from the restrained groups (Fig. 3C and D). Next, equivalent input current (110 pA) was applied to the neurons to avoid the possible influences by the input current intensity. A similar result was found i.e. that the increase in firing frequency by IBTX was greater in cells from the naïve animals than in those from the restrained groups (Fig. 3E and F). These results imply the down-regulation of BKCa channels in cells from the restrained mice.

Figure 3. Reduction in BKCa channel activities after stress.

Figure 3

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A, representative traces showing the firing property in the neurons from LA of naïve and restrained mice response to a 100 pA depolarizing current injection (500 ms). B, the firing frequency to the same input current increased significantly in cells from the single (n = 9 neurons from 3 mice) or two restraints (n = 11 neurons from 3 mice) mice than in cells from the naïve animals (n = 12 neurons from 4 mice). C, representative traces showing the firing property in the neurons from LA of naïve and restrained mice in the presence of IBTX or not. Trains of action potential evoked in the pyramidal neurons in response to a long (500 ms), 100–150 pA depolarizing current injection. D, in the presence of IBTX (100 nm), firing-frequency increase was shown to be greater in cells from the naïve animals (n = 12 neurons from 3 mice) than in cells from the single (n = 10 neurons from 3 mice) or two restraints (n = 14 neurons from 3 mice) groups. E, representative traces showing the firing property in the neurons from LA of naïve and restrained mice in the presence of IBTX or not. Trains of action potentials evoked in the pyramidal neurons in response to a long (500 ms), 110 pA depolarizing current injection. F, in the presence of IBTX (100 nm), firing-frequency increase was shown to be greater in cells from the naïve animals (n = 10 neurons from 4 mice) than in cells from the single (n = 11 neurons from 4 mice) or two restraints (n = 9 neurons from 4 mice) groups. *P < 0.05, **P < 0.01 versus the control.

Western blot analysis was then performed to test the effects of acute stress on expression of BKCa channels in the amygdala. Consistent with the electrophysiological recordings, BKCa channel expression in the amygdala of mice subjected to restraints significantly diminished (Fig. 4A and B). Furthermore, whole-cell recordings revealed a significant decrease in the amplitude of fAHP (Fig. 4C and D) that caused an increase in AP half-width (Fig. 4E) in the amygdala neurons from the mice subjected to restraints.

Figure 4. Down-regulation of BKCa channels by stress.

Figure 4

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A, representative Western blots of BKCa channels expression in total homogenates of the amygdala from the control and restrained mice. B, restraint stress induced a significant reduction of expression levels of the BKCa channels in amygdala of mice subjected to single (n = 8) or two restraints (n = 8) as compared to the naïve mice (n = 8). C, representative recording of the first spike in the neurons from naïve mice subjected to single and two restraints. First action potentials have been superimposed to show the extent of spike broadening and fAHP. The fAHP was defined as the most negative potential after each AP. D, current-clamp recordings revealed a significant decrease in the amplitude of fAHP following the first two spikes in the mice subjected to single (n = 12 neurons from 4 mice) or two restraints (n = 14 neurons from 4 mice) compared with the naïve controls (n = 10 neurons from 5 mice). E, the half-width of the first two spikes was significantly increased in the mice subjected to single (n = 12 neurons from 4 mice) or two restraints (n = 14 neurons from 4 mice) compared with the controls (n = 10 neurons from 5 mice). *P < 0.05, **P < 0.01 versus the control.

Local activation of BKCa channels in the LA reverses stress-induced anxiety-like behaviour

A selective BKCa channel agonist NS1619 was bilaterally infused into the amygdala to directly test the functional involvement of BKCa channels in anxiety-related networks. Pharmacological activation of BKCa channels was expected to reverse stress-induced anxiety-like behaviour in the EPM and open-field tests. Consistent with this, infusion with NS1619 (10 μm, 0.5 μl) reversed the effects of restraints on EPM exploration, measured as time in the open arms, whereas there was no difference in the total number of arm entries between NS1619- and saline-injected animals (Fig. 5A). Similarly, in the open-field test, restrained mice spent more time in the central areas after treatment with NS1619 compared with those injected with saline (Fig. 5B). No significant difference in terms of the total distance travelled within the open field for 30 min was found, indicating that motor coordination and motor function in the mice did not change (Fig. 5B). This demonstrates that pharmacological enhancement of BKCa channel function can reverse the effects of acute stress.

Figure 5. Local infusion of NS1619 reversed stress-induced anxiety-like behaviour.

Figure 5

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A, infusion with BKCa channel agonist NS1619 (10 μm, 0.5 μl) reversed the time in the open arms in Restraint 2 mice, whereas there was no difference in the total number of arm entries between NS1619- (n = 6) and saline-injected animals (n = 8). B, mice infused with NS1619 (n = 6) spent more time in the central areas in open-field test in the Restraint 2 mice compared with saline-treated animals (n = 8). There was no significant difference among the mice in the total distance travelled within the open field for 30 min. *P < 0.05 versus the control.

Enhanced NMDAR function in restrained mice by down-regulation of BKCa channels

Previous research has shown that Ca2+ entry through NMDARs can activate BKCa channels (Isaacson & Murphy, 2001; Shah & Haylett, 2002). It is possible that down-regulation of BKCa by restraints may prolong the repolarization of membrane potential, which would lead to the enhancement of NMDAR activities. Input–output relationships measuring NMDAR-mediated EPSC amplitude (output) as a function of the afferent stimulus intensity (input) were compared in the naïve control and restrained mice at the thalamo-LA synapses to test this hypothesis (Fig. 6A). The slope of the curves was significantly greater in neurons from the restrained mice compared with naïve controls (Fig. 6B), indicating enhanced NMDAR function in the restrained mice. Then, the effects of BKCa channels on NMDAR-mediated EPSPs at the thalamo-LA synapses were tested in current-clamp. NMDAR-EPSPs were elicited using presynaptic stimuli placed on the thalamo-LA fibres (three pulses at 100 Hz) and recorded on the pyramidal neurons in the LA. In the presence of 50 μm CNQX and 100 μm picrotoxin, the traces recorded were NMDAR dependent, as shown by the blockade of the NMDAR blocker AP5 (20 μm) (Fig. 6C). Bath application of IBTX (100 nm) caused an enhancement of NMDAR-EPSPs, whereas activation of BKCa channels with NS1619 (10 μm) induced a significant reduction in NMDAR-EPSPs (Fig. 6D and E).

Figure 6. Effects of BKCa channel on NMDAR-mediated synaptic transmission at thalamo-LA synapse.

Figure 6

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A, diagram showing the placement of stimulating and recording electrodes in the amygdala. B, the NMDAR-mediated EPSCs was pharmacologically isolated in Mg2+-free ACSF containing CNQX (20 μm), glycine (1 μm) and picrotoxin (100 μm). Plot of input–output curves shows significant enhancement of NMDAR-mediated EPSCs amplitude in neurons from restrained mice (n = 12 neurons from 3 restrained mice) compared with naïve controls (n = 10 neurons from 3 mice). C, representative traces showed the effects of bath application of IBTX or NS1619 on total NMDAR-mediated EPSPs in the LA. NMDAR blocker AP5 (20 μm) abolished the EPSP recordedin the LA. Arrowheads indicate the stimulus (three pulses at 100 Hz). D, total NMDAR-mediated EPSPs with the recording time in the control slices (n = 8) and the effects of IBTX (n = 9) or NS1619 (n = 10) on total NMDAR-mediated EPSPs. E, summary of the effects of IBTX and NS1619 on NMDAR-mediated EPSPs in the LA after 15 min perfusion. **P < 0.01 versus the control. F, representative traces showed the effects of bath application of IBTX or NS1619 on total NMDAR-mediated EPSCs in the LA. G, total NMDAR-mediated EPSCs with the recording time in the control slices (n = 8) and the effects of IBTX (n = 8) or NS1619 (n = 7) on total NMDAR-mediated EPSCs. H, summary of the effects of IBTX and NS1619 on NMDAR-mediated EPSCs in the LA after 15 min perfusion.

The hypothesis that the BKCa channel affects membrane voltage to control NMDA receptor activation is reasonable. Thus, under voltage-clamp recordings NMDAR-dependent EPSC amplitude should be unchanged. As shown in Fig. 6F–H, NMDAR-dependent EPSC amplitude was unchanged after bath application of IBTX (100 nm) or NS1619 (10 μm). These results indicate that BKCa channels are functionally linked to the activation of NMDARs.

Changes of the conductance of BKCa channels might directly alter the glutamatergic synaptic transmission in thalamo-LA synapses. As shown in Fig. 7, iberotoxin (100 nm) or NS1619 (10 μm) did not alter the basal synaptic transmission over the entire recording period. No significant alteration was detected in the AMPA receptor-mediated mEPSC frequency and amplitude among the groups (Fig. 7A–C). Furthermore, analysis of the mEPSC kinetics showed no difference in the rising time and decay time among the control, iberotoxin- and NS1619-superfused slices (Fig. 7D). These data indicate that BKCa channels have no significant effects on the basal excitatory synaptic transmission in the lateral amygdala. The alteration of the BKCa channels directly change the strength of NMDAR activity by a postsynaptic mechanism.

Figure 7. Unchanged probability of transmitter release by BKCa channel.

Figure 7

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A, AMPA receptor-mediated mEPSCs recorded in LA pyramidal neurons at a holding potential of –30 mV. Representative traces showed AMPA receptor-mediated mEPSCs in the control, IBTX- and NS1619-incubated slices. B, cumulative frequency (left) and amplitude (right) histogram of the mEPSCs from the cells in A. Continuous line, recording from a control slice; dotted line, recording from an IBTX-incubated slice; dashed line, recording from a NS1619-incubated slice. C, mEPSCs frequency (left) and amplitude (right) in neurons from control (n = 9), IBTX- (n = 12), and NS1619- (n = 10)incubated slices. D, left: average mEPSC of 87 events from a control slice, 92 events from an IBTX-incubated slice, and 78 events from an NS1619-incubated slice. Right: time constant of mEPSC decay (τ) versus the rising time (10–90%) in the recordings from control (n = 9), IBTX- (n = 12) and NS1619- (n = 10) incubated slices.

Discussion

The current study provides four novel findings: (1) acute restraint stress induces anxiety accompanied by down-regulation of BKCa channels in the LA; (2) local activation of BKCa channels in the LA in vivo can reverse anxiety-like behaviour in mice after acute stress; (3) BKCa channels interact with NMDARs in the amygdala; and (4) down-regulation of BKCa channels by acute stress may cause enhanced NMDAR activity at the thalamo-LA synapses. These reflect a novel mechanism that may play an important role in the pathogenesis and therapeutic prevention of stress-induced psychiatric diseases.

Changes in BKCa channels represent a key step in the pathogenesis of acute stress-induced anxiety

The amygdala is critical for processing various kinds of emotions, including fear and anxiety (Fanselow & LeDoux, 1999). Prolonged, intense stress can cause functional and morphological changes in the brain (McEwen, 1999) and trigger pathological anxiety (Pawlak et al. 2003; Rosenkranz et al. 2010). The present study demonstrates that acute stress from one or two sessions of a 2 h restraint is able to induce enhanced anxiety in healthy adult mice. More importantly, this short-term restraint stress leads to a significant down-regulation of BKCa channels in the LA. Stress has been shown to cause neuronal remodelling and modification of synaptic plasticity within the amygdala (Vyas et al. 2002; Maroun & Richter-Levin, 2003). Considering that synaptic integration is critically regulated by the concomitant activation of numerous voltage-gated ion channels in the dendritic membrane (Johnston et al. 2003; Faber et al. 2005), that learning or seizure modulates BKCa channel-mediated fAHP (Matthews et al. 2008; Pacheco Otalora et al. 2008), the BKCa channel possibly plays a role in stress-induced anxiety. Local infusion of drugs activating BKCa channels in the amygdala can reverse this anxiety, pointing to the functional relevance of BKCa channels in the anxiety-related network of the amygdala. These data are the first to our knowledge to demonstrate that, in addition to other changes, alterations in BKCa channels in the LA play an important role in the pathogenesis of anxiety after stress. Furthermore, they suggest that down-regulated BKCa channels represent one of the key steps of stress-induced changes that may trigger further pathophysiological processes.

BKCa channel and neuronal excitability

Activation of BKCa channels is critically dependent on the AP to which BKCa channels contribute AP repolarization and fAHP (Lancaster et al. 1991; Faber & Sah, 2003; Haghdoost-Yazdi et al. 2008; Womack et al. 2009). BKCa-dependent fAHP is brief (< 20 ms) (Sah, 1996; Sah & Faber, 2002) but often affects the firing rate or pattern in some cells (Sah, 1996; Poolos & Johnston, 1999; Martinez-Pinna et al. 2000; Lovell & McCobb, 2001; Smith et al. 2002). The present study provides direct evidence of the contribution of BKCa channels to the excitability of pyramidal neurons in the amygdala. Blockade of the BKCa led to an increase in the firing number and a notable reduction in the negative-going peak of the fAHP. In addition, alterations in fAHP and AP half-width by BKCa channels were occluded in the neurons of mice after stress. These results demonstrate that blockade of BKCa channels and subsequent changes in the shapes of AP and fAHP disturb the function of the neuronal network in amygdala, thus enhancing anxiety. These data further point to the functional importance of BKCa channels in the pathogenesis of psychiatric diseases.

Functional coupling between BKCa channels and NMDARs

Investigations of BKCa channels in various types of neurons show that activation of BKCa channels requires the delivery of Ca2+ through voltage-dependent calcium channels (Cav channels) because blocking these channels inhibits BKCa-mediated currents (Prakriya & Lingle, 1999; Edgerton & Reinhart, 2003; Fakler & Adelman, 2008). The greater ability of BAPTA compared with EGTA to interfere with BKCa channel gating strongly suggests nanometer distances between BKCa and Cav channels (Muller et al. 2007; Fakler & Adelman, 2008). Ca2+ entry exclusively through NMDARs has been reported to activate BKCa (Isaacson & Murphy, 2001; Shah & Haylett, 2002) and SK (Faber et al. 2005; Ngo-Anh et al. 2005) channels. From the present study, we provide strong electrophysiological evidence to show that BKCa channels are functionally coupled to NMDA receptors, even though BKCa channels may functionally couple to other receptors, such as voltage-dependent calcium channels, through which calcium influxes into the cells. This functional coupling between BKCa and NMDA receptors contributes to altered excitability of neurons under acute stress for the down-regulation of BKCa.

Many studies support the notion that the LA is an essential site where NMDAR-dependent changes in neuronal activity are required for the acquisition of conditioned fear and in assigning stress disorders (Quirk et al. 1995; Gewirtz & Davis, 1997; Pawlak et al. 2003; Ehrlich et al. 2009). This has led to the conclusion that alterations in BKCa channels and NMDARs at sensory afferents to the LA projection neurons underlie this process. The present study shows that blockade of BKCa channels significantly enhances NMDAR-mediated transmission and that activation of BKCa channels leads to shunting of NMDAR-mediated EPSPs at the thalamo-LA synapses; in addition, it identifies BKCa channels in the amygdala as a key player in a sequence of events that may link experience-dependent plasticity with the development of anxiety after stress.

The present study provides a novel mechanism for evaluating the effects of stress on emotions. Changes in BKCa channels may represent one of the key steps of stress-induced changes in the neuronal network of amygdala. These initial changes can trigger other pathophysiological processes. Detailed knowledge of these mechanisms will help in better understanding of stress-induced pathophysiological processes and in developing therapeutic interventions for preventing anxiety, depression, or post-traumatic stress disorder.

Acknowledgments

The authors thanked Dr Min Zhuo (University of Toronto) and Dr Yutian Wang (University of British Columbia) for their constructive discussion for this manuscript. This work was supported by National Natural Science Foundation of China No. 30770686, 31070923, 2008 ZXJ09004-023 and 2009ZX09103-111, and Program for New Century Excellent Talents in University.

Glossary

Abbreviations

AP

action potential

BKCa

large-conductance Ca2+-activated potassium channel

EPM

elevated plus maze

EPSC

excitatory postsynaptic current

EPSP

excitatory postsynaptic potential

fAHP

fast after-hyperpolarization

LA

lateral amygdala

OP

open field

Author contributions

Y.-y.G. YY, S.-b.L., G.-B.C., L.M., B.F. and J.-h.X. performed the experiments. Q.Y., X.-q.L. and Y.-m.W. analysed the data. L.-z.X., W.Z. and M.-g.Z. designed the research and wrote the manuscript. All authors approved the final version for publication, and the work was performed at the Fourth Military Medical University.

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