Acetylcholine (ACh) is an important modulator of learning, memory, and synaptic plasticity in the basolateral amygdala (BLA) and other brain regions. Activation of muscarinic acetylcholine receptors (mAChRs) suppresses a variety of potassium currents, including sIAHP, the calcium-activated potassium conductance primarily responsible for the slow afterhyperpolarization (AHP) that follows a train of action potentials. Muscarinic stimulation also produces inositol 1,4,5-trisphosphate (IP3), releasing calcium from intracellular stores. Here, we show using whole-cell patch-clamp recordings and high-speed fluorescence imaging that focal application of mAChR agonists evokes large rises in cytosolic calcium in the soma and proximal dendrites in rat BLA projection neurons that are often associated with activation of an outward current that hyperpolarizes the cell. This hyperpolarization results from activation of small conductance calcium-activated potassium (SK) channels, secondary to the release of calcium from intracellular stores. Unlike bath application of cholinergic agonists, which always suppressed the AHP, focal application of ACh often evoked a paradoxical enhancement of the AHP and spike-frequency adaptation. This enhancement was correlated with amplification of the action potential-evoked calcium response and resulted from the activation of SK channels. When SK channels were blocked, cholinergic stimulation always reduced the AHP and spike-frequency adaptation. Conversely, suppression of the sIAHP by the β-adrenoreceptor agonist, isoprenaline, potentiated the cholinergic enhancement of the AHP. These results suggest that competition between cholinergic suppression of the sIAHP and cholinergic activation of the SK channels shapes the AHP and spike-frequency adaptation.
The amygdaloid complex is a collection of deep temporal lobe nuclei involved in emotion and emotional learning. Within this complex, neurons in the basolateral amygdala (BLA) are proposed to be involved in assigning affective value to sensory stimuli. This association results in a change in the output [action potentials (APs)] of BLA projection neurons to the sensory stimulus. Functionally, neuronal output is determined by interactions between synaptic inputs impinging on neurons and their intrinsic excitability. Intracellular recordings have shown that BLA projection neurons in vivo discharge high-frequency bursts both spontaneously (Pare and Gaudreau, 1996) and in response to depolarizing current injections (Pare et al., 1995). Activation of calcium-activated potassium channels has been implicated in the generation of this bursting behavior (Lang and Pare, 1997; Chen and Lang, 2003). Similarly, the strongly adapting firing pattern observed in vitro is attributable to, in large part, the pronounced slow afterhyperpolarization (AHP) that follows trains of APs (Washburn and Moises, 1992a; Rainnie et al., 1993).
As in many other neurons, the AHP is primarily mediated by two calcium-activated potassium currents, IAHP and the sIAHP (Sah and Faber, 2002), which are activated by calcium influx during action potentials. The IAHP current is mediated by small conductance calcium-activated potassium (SK) channels, and its time course primarily follows cytosolic calcium, rising rapidly after APs and decaying with a time constant of 50 to several hundred milliseconds (Sah and Faber, 2002). In contrast, the kinetics of the sIAHP are slower, exhibiting a distinct rising phase and decaying with a time constant of 1–2 s (Sah, 1996). A variety of neuromodulators, including acetylcholine (ACh), noradrenaline, and glutamate acting via G-protein-coupled receptors, suppress the sIAHP and thus reduce spike-frequency adaptation (Nicoll, 1988).
The BLA receives one of the densest cholinergic innervations in the CNS (Ben-Ari et al., 1977; Mash and Potter, 1986). Consistent with this, neurons in the BLA express a variety of muscarinic (Levey et al., 1991) and nicotinic (Zhu et al., 2005) receptors. Although the precise mechanism of action is unclear, activation of cholinergic receptors have been implicated in amygdala-dependent learning (McGaugh, 2004; Tinsley et al., 2004; Barros et al., 2005). They are also known to modulate long-term synaptic plasticity within the amygdala (Watanabe et al., 1995). Physiologically, activation of muscarinic receptors on BLA neurons suppresses several potassium currents, including the sIAHP, as well as the voltage-sensitive M-current (Washburn and Moises, 1992b; Womble and Moises, 1993). These receptors also activate a nonspecific cation conductance (Yajeya et al., 1999). All these actions enhance neuronal excitability and are thought to underlie the cellular actions of acetylcholine. However, muscarinic stimulation also generates inositol 1,4,5-trisphosphate (IP3) that releases calcium from intracellular stores, a process that is amplified by AP-evoked calcium influx (Power and Sah, 2005, 2007). Store-released calcium activates SK calcium-activated potassium channels in midbrain dopamine neurons (Fiorillo and Williams, 1998, 2000; Morikawa et al., 2003) and cortical pyramidal neurons (Yamada et al., 2004; Gulledge and Stuart, 2005; Hagenston et al., 2007), consequently modulating neuronal excitability.
In this study, we examine the roles of calcium-activated potassium channels in modulating the firing properties of basal amygdala projection neurons. We show that, in these neurons, the AHP is mediated by a combination of apamin-sensitive SK channels and the apamin-insensitive sIAHP. Calcium release from intracellular stores, initiated by activation of muscarinic receptors, activates somatic SK channels and can enhance the AHP under some circumstances. These results show that muscarinic activation of SK channels competes with muscarinic suppression of the sIAHP to shape the AHP and spike-frequency adaptation.
Materials and Methods
Coronal brain slices (350–400 μm) were prepared using standard techniques (Power and Sah, 2002). Wistar rats (21–28 d of age) were anesthetized with halothane and decapitated. Slices were prepared using a vibrating microslicer (DTK-1000; Dosaka, Kyoto, Japan). These procedures were conducted in accordance with the guidelines of the University of Queensland Animal Ethics Committee. Slices were incubated at 33°C for 30 min and then maintained at room temperature in artificial CSF (ACSF) solution containing the following (in mm): 119 NaCl, 2.5 KCl, 1.3 MgCl2, 2.5 CaCl2, 1.0 Na2H2PO4, 26.2 NaHCO3, 11 glucose, equilibrated with 95% CO2, 5% O2. Slices were perfused with ACSF heated to 33°C, and whole-cell recordings were made from the soma of BLA neurons using infrared differential interference contrast video microscopy. Patch pipettes (2–5 MΩ) were filled with an internal solution containing the following (in mm): 135 KMeSO4, 8 NaCl, 10 HEPES, 2 Mg2-ATP, 0.3 Na3-GTP, 0.1 spermine (pH 7.3 with KOH, osmolarity 280–290 mOsm). Electrophysiological signals were amplified with either an Axopatch 1D or a Multiclamp 700A amplifier (Molecular Devices, Union City, CA), filtered at 2–5 kHz and digitized at 5–10 kHz with an ITC-16 board (Instrutech, Port Washington, NY), and controlled using Axograph 4.9 (AxoGraph Scientific, Sydney, Australia). Whole-cell recordings were obtained from projection neurons in the basal nucleus of the BLA. Only cells that had resting potentials more negative than −55 mV, AP amplitudes >100 mV, and membrane resistances (Rm) > 60 MΩ were included in the data set. Projection neurons were distinguished from local circuit interneurons based on the brevity of their action potentials (>0.7 ms), fast AHP (<15 mV), and their frequency-dependent spike broadening.
Whole-field fluorescence measurements were made using a monochromator-based imaging system, Polychrome II (T.I.L.L. Photonics, Munich, Germany) with 50 μm Oregon Green BAPTA-1 (Invitrogen, San Diego, CA) included in the internal solution. Neurons were visualized using a BX50 microscope (Olympus Optical, Tokyo, Japan) equipped with a 60× water immersion objective (numerical aperture, 0.9; Olympus) and illuminated with 488 nm light. Images were acquired with an interline transfer, cooled CCD camera (T.I.L.L. Photonics) in which the scan lines were binned by two in both horizontal and vertical directions, giving a spatial resolution of 0.33 μm per pixel. Frames were collected at 25–33 Hz with a 10 ms exposure time. Images were analyzed off-line using Vision (T.I.L.L Photonics). Calcium signals were calculated as the relative change in fluorescence over baseline fluorescence (ΔF/F). ΔF/F(t) = [F(t) − F0]/(F0 − B), where Ft is the fluorescence at time t, F0 is the average baseline fluorescence before the stimulus, and B is the background fluorescence measured in an adjacent extracellular region.
Two-photon fluorescence images were obtained using a Zeiss (Oberkochen, Germany) Axioskop 2FS with a 510 laser scanning head equipped with a Chameleon laser (Coherent, Santa Clara, CA) for two-photon excitation. For two-photon experiments, the green fluorescent calcium indicator Fluo 5F (300 μm; Kd, 2 μm; Invitrogen, Carlsbad, CA) was added to the internal solution along with the red fluorescent calcium-insensitive Alexa 594 (30 μm; Invitrogen). Fluo 5F and Alexa 594 were excited at 810 nm. The emitted light was split with a dichroic (DT560), bandpass filtered (green channel, 500–560 nm; red channel, 575–640 nm), and detected with separate nondescanned detectors. Fluorescence images were acquired in line scan-mode (100–500 Hz) at a resolution of 10–20 pixels μm−1. Small segments were selected over each subcellular region, and the fluorescence over this area was averaged. For two-photon sequences, calcium signals were calculated as the change in green fluorescence (Fluo 5F) normalized to the red fluorescence (Alexa 594), ΔG/R(t). Calcium signals were analyzed off-line using custom software written using LabVIEW (National Instruments, Austin, TX).
For focal application of cholinergic agonists, ACh or muscarine were loaded into a patch pipette and applied using either a picospritzer for pressure application (10–30 psi, 50–500 ms; Parker Hannifin Fairfield, NJ) or the 700A amplifier for iontophoretic application. For pressure application, drugs were dissolved in ACSF at a concentration of 10–20 μm. For iontophoretic application, acetylcholine (10–25 mm; pH 4 with HCl) was applied with a 2–50 nA ejection current and retained with a −5 nA retention current. The metabotropic glutamate receptor agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) (2 mm; pH 8.5), was applied using a −50 nA ejection current and a 10 nA retention current. All other drugs were bath applied. Cyclopiazonic acid (CPA), 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl] (phenylmethyl) phosphinic acid (CGP 55845) were prepared as stock solutions in DMSO and diluted in ACSF when required. NBQX, CGP 55845, and 2-amino-5-phosphonovaleric acid (APV) were purchased from Tocris Bioscience (Bristol, UK). All other drugs were obtained from Sigma-Aldrich (St. Louis, MO). Drugs were prepared as 1000× stock solutions and stored frozen (−20°C) until required.
Depletion of intracellular calcium stores was prevented by maintaining the neuron at a slightly depolarized membrane potential (Vm, −50 mV) or evoking AP between ACh applications (Power and Sah, 2005). The AHP was evoked by a 100 Hz train of 5–10 ms depolarizing current injections, where each current step evoked a single AP. Occasionally two trains (200 ms intertrain interval) of depolarizing current injections were given to evoke both a robust AHP and calcium response. Measurements of the AHP included the peak amplitude, integrated area, mean afterpotential (0–5 s after the last current step), and the amplitude of the AHP 100 ms, 1 s, 2 s, and 5 s after the last current step. The peak and integrated area refer to negative (hyperpolarized) values only. Calcium response measures included the peak amplitude, integrated area, and the mean response 0–5 s after the last current step. For experiments using linopiridine the AHP and spike-frequency adaptation were measured alternatively at 1 min intervals before and during application of channel blockers. Inhibition was considered at steady state levels 7–10 min after the onset of drug application. AHP measurements were made from the average of two to three responses immediately before drug application and after blockers reached steady-state levels. Spike-frequency adaptation was measured from each current injection, and the responses were averaged. Statistical comparisons were made using a paired Student's t test, unless otherwise indicated. All data are presented as mean ± SEM unless otherwise indicated.
Whole-cell patch-clamp recordings were made from projection neurons within the basal nucleus of the BLA. These neurons had resting potentials of −65.0 ± 4.3 mV (mean ± SD) and input resistances of 120 ± 66 MΩ (mean ± SD; n = 154). Projection neurons in the basal nucleus are traditionally separated into two classes based on their firing patterns in response to depolarizing current injections (Washburn and Moises, 1992a), although it is unclear whether these two classes represent distinct neuronal populations or are on opposite ends of a firing property continuum (Kroner et al., 2005). The majority of neurons (80%) had a pyramidal-like firing pattern with strong spike-frequency adaptation and a prominent AHP (Fig. 1A,B). Less frequently (20%), we encountered neurons that showed a late-firing phenotype where the onset of spike firing was delayed (>300 ms) near threshold (Fig. 1A). As described previously (Washburn and Moises, 1992a), late-firing neurons tended to show less spike-frequency adaptation than pyramidal-like neurons, and their AHP was generally smaller in amplitude (Fig. 1A,B, middle). However, nearly half of the late-firing neurons we encountered exhibited robust spike-frequency adaptation and pronounced AHPs (Fig. 1A,B, right). The AHP amplitude was 2.0 ± 0.5 mV for late-firing neurons (n = 17) compared with 2.7 ± 0.2 mV for pyramidal-like neurons (n = 48; p = 0.18). It has been reported that late-firing neurons are more hyperpolarized than pyramidal-like neurons, and the kinetics of their AHP differs from that in pyramidal-like neurons (Washburn and Moises, 1992a; but see Kroner et al., 2005). We did not observe any differences in either their resting membrane potential (late-firing neurons, −65.6 ± 0.8 mV; pyramidal-like neurons, −64.8 ± 0.4 mV; p = 0.35) or the membrane resistance (late-firing neurons, 137 ± 9 MΩ; pyramidal-like neurons, 115 ± 7 MΩ; p = 0.11) between classes. Although the AHP that followed a brief AP train was generally smaller in amplitude for late-firing neurons that did not accommodate, we found that both the calcium responses (data not shown) and the AHP time courses (Fig. 1B) were indistinguishable between these two types of cells, and the data have been combined. The AHP showed slow kinetics in both cell types with 32 ± 7 and 37 ± 2% of the AHP amplitude remaining 2 s after the pulse train for late-firing (n = 17) and pyramidal-like (n = 48) neurons, respectively (p = 0.38). To limit variability, however, spike-frequency adaptation was examined solely in pyramidal-like neurons.
In most neurons, the AHP is primarily mediated by either the apamin-sensitive IAHP or the apamin-insensitive sIAHP and, in many cases, a combination of these two currents (Sah and Faber, 2002). It was initially reported that the apamin-sensitive IAHP is not present in BLA projection neurons (Womble and Moises, 1993). However, subsequent reports have indicated that SK calcium-activated potassium channels, which mediate the IAHP, are present in projection neurons in the lateral nucleus of the BLA (Faber and Sah, 2002; Chen and Lang, 2003; Faber et al., 2006). To determine whether the SK channels (IAHP) contribute to the AHP in projection neurons in the basal nucleus, we examined the AHP evoked by four APs (100 Hz) before and after SK channel blockade by bath application of either apamin (100 nm) or 8,14-diaza-1,7(1, 4)-diquinolinacyclotetradecaphane (UCL 1848; 1 μm) (Chen et al., 2000).
Application of specific SK channel blockers had a modest but significant effect on the AHP, reducing its peak by 28 ± 9% (p = 0.020; n = 7) and its integrated area by 17 ± 8% (p = 0.068; n = 7) (Fig. 1C). Consistent with the relatively rapid kinetics of the IAHP (Sah and Faber, 2002), the effect of SK channel blockade was greatest shortly after the spike train but produced only a modest reduction (19 ± 7%; p = 0.033; n = 7) in the AHP amplitude measured 1 s after the spike train (Fig. 1C). In response to a depolarizing current injection, pyramidal-like neurons fire a burst of APs, which rapidly accommodates. Blockade of SK channels produced a modest attenuation of this spike-frequency adaptation (Fig. 1C3), increasing the mean AP frequency evoked by a 600 ms, 400 pA current injection from 4.8 ± 0.8 to 6.1 ± 1.0 Hz (p = 0.023; n = 9).
Consistent with previous reports (Womble and Moises, 1993), suppression of the sIAHP by bath application of muscarine (5–20 μm) resulted in a reduction in both the AHP and spike-frequency adaptation (Fig. 1D). Muscarine reduced the peak amplitude of the AHP by 84 ± 7% (p = 0.00052), the integrated area by 94 ± 3% (p = 0.00546), and completely abolished the AHP measured 1 s after the AP train (100 ± 0% block; p = 0.00036). In many instances, the latter portion of the AHP was replaced by a slow afterdepolarization (Fig. 1D). The result was an increase in the mean spike frequency during a 600 ms, 400 pA current injection from 4.4 ± 0.8 to 7.5 ± 1.6 Hz (p = 0.047; n = 8).
In addition to the sIAHP, muscarinic stimulation also suppresses the voltage-sensitive M-current, which has been shown to modulate the AHP that contributes to spike-frequency adaptation in other neuronal types (Gu et al., 2005). To determine whether the M-current contributes to the AHP and spike-frequency adaptation, we applied the specific M-current blocker linopiridine (Schnee and Brown, 1998). Linopirdine (10 μm) had no effect on the AHP to a brief four-AP train (Fig. 1E). The peak amplitude and area of the AHP in the presence of linopirdine were 87 ± 11% (p = 0.43) and 101 ± 18% (p = 0.64) of control, respectively (n = 5). In BLA neurons, the sIAHP and the M-current are also suppressed by metabotropic glutamate receptor (mGluR) activation (Womble and Moises, 1994). In the presence of linopirdine, application of the mGluR agonist t-ACPD suppressed the AHP (Fig. 1E) (n = 2), indicating that muscarinic suppression of the AHP results from blockade of the sIAHP rather than the suppression of the M-current. This current does, however, play a role in spike frequency adaptation as linopirdine increased the average AP frequency during a 400 pA, 600 ms current step from 6.0 ± 1.1 to 8.2 ± 1.2 Hz (p = 0.034; n = 5). Thus, in projection neurons in the basal nucleus, both IAHP and sIAHP contribute to the AHP with the apamin-insensitive sIAHP making the greatest contribution (Fig. 1F). The M-current, although present, is not active during the AHP but does affect spike-frequency adaptation.
Release of calcium from intracellular stores activates SK channels
Brief focal application of ACh or muscarine evokes large (>1 μm) wave-like rises in intracellular calcium in the soma and proximal dendrites that result from release of calcium from IP3-sensitive intracellular calcium stores (Power and Sah, 2007). Interestingly, although release of calcium from IP3-sensitive intracellular calcium stores can also be observed in response to bath application of muscarinic agonists, focal stimulation is much more effective in evoking robust calcium rises (Power and Sah, 2002, 2007). This difference may result from desensitization of muscarinic receptors as receptor occupancy rises slowly during bath application. Focally evoked calcium waves were frequently associated with an outward current in voltage-clamp mode (Fig. 2A) or with membrane hyperpolarization with the cell in current clamp. Both the evoked calcium rise and outward current were only seen in response to agonist application onto the soma or proximal dendrites and were not observed (0 of 4) when agonists were applied to dendrites further than 60 μm from the soma (Power and Sah, 2007).
To eliminate movement artifacts and minimize problems related to calcium indicator saturation, neurons were focally stimulated by iontophoretic application of ACh and imaged using multiphoton fluorescence microscopy and the moderate affinity calcium indicator Fluo 5F (Kd, ∼2 μm). Because ACh also activates nicotinic receptors in these neurons (Klein and Yakel, 2006), experiments using ACh were performed in the presence of the nicotinic receptor antagonist mecamylamine (10 μm). The outward current evoked by ACh was blocked by atropine (1 μm, n = 3) (Fig. 2B) and was not affected by application of GABAergic (50 μm picrotoxin and 10 μm CGP 58845) and ionotropic glutamatergic blockers (2 mm kynurenic acid or 30–60 μm APV plus 20 μm NBQX), suggesting that it results from the direct actions of mAChR activation on BLA projection neurons rather than network effects. Moreover, the outward current was reversibly blocked by CPA, which blocks the endoplasmic reticulum (ER) Ca2+ ATPase and empties intracellular calcium stores (Fig. 2C) (n = 6). CPA (15–30 μm) abolished the cholinergic-evoked calcium rise and reduced the amplitude of the outward current by 82.5 ± 4.0% (p = 0.008) and the area of the outward current by 96.1 ± 1.8% (p = 0.022). On occasion, muscarinic stimulation evoked a small inward current with slow kinetics (Fig. 2C,D). This current was not blocked by CPA and, although we have not characterized it fully, its properties are consistent with activation of a nonspecific cationic conductance by muscarinic receptor stimulation (Yajeya et al., 1999). Release of calcium from intracellular stores is dependent on store content, with repetitive activation decreasing the store content and reducing release from intracellular calcium stores (Power and Sah, 2005). Conversely, calcium entry through voltage-gated calcium channels, during APs or depolarizations near threshold, primes the calcium stores, augmenting the calcium response to cholinergic stimulation (Power and Sah, 2005). Thus, when neurons were held at −65 mV, repeated application of ACh reduced both calcium store release and the outward current (Fig. 2D). Replenishing the calcium store with a 1 min membrane depolarization (−50 mV) augmented both store release and the outward current (Fig. 2D). We have shown previously that when cholinergic agonists are applied to the proximal dendrite, the calcium rise begins as a focal rise in the dendrite, which propagates into the soma (Power and Sah, 2007). Inspection of the timing of calcium rises in the soma and dendrite reveals that the onset of the outward current is best correlated with the somatic calcium rise (Fig. 2E), suggesting that the channels that underlie this current have a somatic location.
Calcium release from internal stores has been found to activate SK channels in other neurons (Fiorillo and Williams, 1998, 2000; Morikawa et al., 2003; Yamada et al., 2004; Gulledge and Stuart, 2005; Hagenston et al., 2007). Consistent with this mechanism, the outward current activated by muscarinic agonists in BLA neurons was suppressed by bath application of the SK channel blocker apamin (100 nm), with no effect on the calcium rise. Apamin reduced the amplitude of the outward current by 78.4 ± 3.2% (p = 0.006) and the area by 94.1 ± 0.3% (p = 0.025) (Fig. 2F) (n = 4), whereas the peak amplitude and the area of the calcium rise were unaffected (95.7 ± 3.5% of control; p = 0.33 and 97.0 ± 10.7% of control; p = 0.51, respectively). Because muscarinic receptors activate a second messenger cascade, we next examined whether SK channels could also be directly modulated by ACh. In the presence of CPA to block store release, we evoked an AHP current via a depolarizing voltage step (500 ms, 70 mV; Vm, −50 mV) with and without iontophoretic application of ACh (2 s, 50 nA). We subsequently evoked the AHP current with and without ACh in the presence of bath applied apamin. Subtracting the AHP currents evoked in apamin from their respective control currents, we were able to determine the apamin-sensitive (SK) current with and without application of ACh. When store release was blocked, iontophoretic application of ACh had no effect on the integrated area of the apamin-sensitive current (103 ± 3% of control; p = 0.47; n = 4) (Fig. 2G). Thus, muscarinic receptor activation does not directly modulate SK channels. These results, together with the effects of store depletion and priming, indicate that in BLA projection neurons, cholinergic stimulation releases calcium from intracellular stores, which in turn activates SK potassium channels.
In BLA projection neurons (Power and Sah, 2007) and elsewhere (Jaffe and Brown, 1994; Nakamura et al., 1999; Ross et al., 2005), calcium waves can also be evoked by exogenous application of group I mGluR agonists and synaptically through the cooperative action of mGluR and mAChR stimulation (Power and Sah, 2007). Thus, as with muscarinic stimulation, focal application of the mGluR agonist t-ACPD evoked a rise in somatic calcium and an outward current (Fig. 3A,C). As with muscarinic stimulation, apamin reduced the peak of the t-ACPD evoked current by 82 ± 4% (p = 0.010) and the area by 94 ± 4% (p = 0.030) without affecting the somatic calcium rise (peak, 89 ± 13%, p = 0.35; area, 98 ± 12%, p = 0.49; n = 4). Moreover, the outward current was not detected before the somatic calcium rise (Fig. 3B), regardless of whether the wave is initiated in the dendrite or the soma. Similarly, when calcium waves were evoked by synaptic stimulation (in the presence of GABAergic and iontotropic glutamatergic blockers) (Fig. 3D), somatic invasion of the calcium wave was usually (21 of 24 neurons) associated with an outward current. Muscarinic receptors are thought to play a critical role in somatic invasion of synaptically evoked calcium waves (Watanabe et al., 2006; Power and Sah, 2007). Application of atropine reduced the integrated area of the synaptically evoked somatic calcium rise by 82 ± 9% (p = 0.004) and the outward current by 83 ± 17% (p = 0.02; n = 4) (Fig. 3E,F), indicating that much of the somatic calcium rise and outward current are caused by mAChR activation. This outward current resulted from SK channel activation, because application of apamin reduced the amplitude and area of the synaptically activated outward current by 82 ± 8% (p = 0.006) and 96 ± 3% (p = 0.04), respectively (n = 5) (Fig. 3G–I). The peak amplitude and the area of the calcium rise were unaffected by apamin (118 ± 12% of control, p = 0.23, and 133 ± 20% of control, p = 0.26, respectively). Thus, regardless of how calcium waves were evoked (ACh, t-ACPD, synaptically), an apamin-sensitive outward current is activated with a time course that correlates well with somatic calcium rises. This is despite the fact that the calcium rise is largest in proximal dendrites (10–20 μm from the soma) typically exceeding 1 μm (Power and Sah, 2007). Given that the EC50 value for SK channels is 300–700 nm (Stocker, 2004), it seems unlikely that these SK channels are present on the proximal dendrite but are activated by calcium released from intracellular stores that are located on or near the soma.
Competing effects of ACh on the AHP
We have shown that muscarinic stimulation has two actions. First, it enhances neuronal excitability by reducing the AHP (Fig. 1D). However, activation of muscarinic receptors also causes release of calcium from intracellular calcium stores, which activates SK calcium-activated potassium channels (Fig. 2F). The resultant hyperpolarization would be expected to reduce neuronal excitability. We therefore examined how these competing actions of cholinergic stimulation on calcium-activated potassium currents shape the AHP. We first compared the AHP evoked by an AP train with and without iontophoretic application of ACh. Calcium is a coagonist at IP3 receptors (Bezprozvanny et al., 1991; Finch et al., 1991; Mak et al., 1999) and, in the presence of IP3, AP-evoked calcium influx can be amplified through IP3-assisted calcium-induced calcium release (IP3-CICR) (Taylor and Marshall, 1992; Nakamura et al., 1999; Power and Sah, 2002). An example of IP3-CICR is shown in Figure 4. In BLA projection neurons, AP trains evoke a rapid rise in calcium in the soma and dendrite that decays immediately after the last AP. The AP train also evokes an AHP, which shows a slower time course than the calcium response (Fig. 4A). In the same neurons, focal application of lower levels of ACh (obtained by reducing the duration and amplitude of the ejection current) produces little or no calcium response or corresponding membrane hyperpolarization. Pairing the same level of ACh with an AP train results in a calcium rise in the soma and proximal dendrites that is larger than the arithmetic sum of the separate ACh and AP presentations (Fig. 4B). In some instances, the amplified calcium response was accompanied by a clear enhancement of the AHP (Fig. 4A,B). IP3-CICR is also seen in response to bath application of mAChR agonists (Power and Sah, 2002, 2007), although the amplification is markedly smaller than that observed in response to focal stimulation (Fig. 4C). This amplification is largest in response to the first AP train and then declines, presumably because of desensitization of receptors in the continued presence of agonist (Fig. 4C).
The mean response to focal application of ACh was a slight (0.67 mV) enhancement of the AHP (n = 30), although this response was heterogeneous (Fig. 5). In some neurons focal application of ACh augmented the AHP, resulting in a more negative afterpotential (Figs. 4, 5A), whereas in others, it suppressed the AHP, resulting in a more positive afterpotential (Fig. 5B). A histogram of the distribution of the cholinergic modulation of the AHP peak amplitude is shown in Figure 5C. Because the AHP is a negative potential, enhancement of the AHP is plotted as a negative change in the AHP, whereas suppression of the AHP is plotted as a positive change in the AHP. We found that the variability of the response was attributable to, in part, variations in ACh-evoked store release. Here, the effect of ACh on the AHP was correlated with the extent to which ACh augmented the AP-evoked calcium response (Fig. 5D), with larger calcium responses being associated with an enhanced AHP. There was a clear correlation between the amplitude of the amplified calcium response (measured as the integrated area of the amplified portion of the response) and the change in the peak (r = 0.637; p < 0.0001), and the integrated area (r = 0.541; p = 0.0079), of the AHP as well as the amplitude of the AHP 100 ms (r = 0.710; p < 0.0001), 1 s (r = 0.675; p < 0.0001), and 2 s (r = 0.659; p < 0.0001) after the last AP.
As shown above (Fig. 4), mAChR activation generates an outward current that augments the AHP. This augmentation was blocked by apamin or UCL 1848, showing that the AHP evoked during muscarinic stimulation is primarily caused by activation of SK-type calcium-activated potassium channels (Fig. 6A). Moreover, in neurons where cholinergic stimulation evoked IP3-CICR but produced a net reduction in the AHP, the cholinergic reduction of the AHP was often greater when SK channels were blocked (Fig. 6B). These results show that activation of SK channels via IP3-CICR masks the suppression of the AHP typically seen in response to bath application of mAChR agonists. Cholinergic suppression of the sIAHP occurs through activation of muscarinic receptors linked to phospholipase C (Dutar and Nicoll, 1989; Washburn and Moises, 1992b), possibly through activation of calcium calmodulin-dependent protein kinase II (Muller et al., 1992; Krause and Pedarzani, 2000). Thus, cholinergic phosphoinositol turnover both releases calcium from IP3-sensitive ER calcium stores and suppresses the sIAHP. To isolate the cholinergic action on the AHP and IP3-CICR from its modulation of the sIAHP, we applied the β-adrenergic receptor agonist isoprenaline. Stimulation of β-adrenergic receptors suppresses the sIAHP (Nicoll, 1988; Huang et al., 1994; Faber and Sah, 2005) through protein kinase A and is not linked to stimulation of phospholipase C (Pedarzani and Storm, 1993). When the sIAHP was suppressed by bath application of isoprenaline (10 μm), focal application of ACh always enhanced the AHP, even in neurons where ACh previously suppressed it (Fig. 6C,E). Thus, a competition exists between cholinergic suppression of the sIAHP and cholinergic activation of the SK channels resulting from calcium release from intracellular stores. Blockade of SK channels unmasked the cholinergic suppression of the sIAHP (Fig. 6D), whereas suppression of the sIAHP by isoprenaline unmasked cholinergic activation of SK channels (Fig. 6E). Neither the suppression of the sIAHP nor the blockade of SK channels altered the amplification of the calcium response by ACh. Thus, under conditions of SK channel blockade, there was no longer a relationship between cholinergic augmentation of the calcium response and the AHP (Fig. 6F).
The major role of the slow AHP in neurons is thought be in spike-frequency adaptation (Sah, 1996). We next tested whether neurons that showed an enhanced AHP to focal ACh stimulation would also exhibit enhanced spike-frequency adaptation. AP trains (10 Hz, 10 s) were evoked using 50 ms current injections set 25 pA above threshold. Data were partitioned into 1 s bins, and spike frequency was calculated by counting the number of spikes within each 1 s bin. Under control conditions, the AP firing rate showed marked accommodation (Fig. 7A). In neurons where ACh iontophoresis enhanced the AHP, focal application of ACh resulted in an early transient enhancement of spike-frequency adaptation (reduced firing) that was followed by a prolonged period of reduced spike-frequency adaptation (enhanced firing). This enhanced spike-frequency adaptation was associated with an augmented somatic calcium rise. In contrast, neurons in which ACh suppressed the AHP showed little or no suppression of spike-frequency adaptation (Fig. 7B) and a less pronounced calcium rise (Fig. 7C). The prolonged period of enhanced firing did not differ between groups and presumably resulted from the cholinergic suppression of sIAHP and IM, together with activation of a mixed cation conductance. Blockade of SK channels eliminated the cholinergic enhancement of spike-frequency adaptation during the first two intervals (p values <0.05), without affecting the augmentation of the calcium rise (Fig. 8A–C) (n = 5). The prolonged reduction of spike-frequency adaptation was not affected by application of apamin. Consistent with the enhanced spike-frequency adaptation from SK channel activation being secondary to calcium store release, depletion of calcium stores with CPA blocked both the cholinergic enhancement of the AP evoked calcium response and the cholinergic enhancement of spike frequency adaptation (Fig. 8D–F) (n = 6). CPA significantly reduced the cholinergic suppression of spike-frequency adaptation during the first interval (p < 0.05), along with the cholinergic augmentation of the calcium response in the first five intervals (p values <0.05). Spike-frequency adaptation was reduced when the sIAHP was suppressed by bath application of isoprenaline (Fig. 8G) (n = 6; p values = 0.06–0.01). The early enhancement of spike-frequency adaptation by focal application of ACh was augmented after application of isoprenaline (Fig. 8G–I) (n = 6; interval 1, p < 0.05). Together, these results indicate that ACh modulates spike-frequency adaptation through the opposing actions of sIAHP suppression and SK channel activation.
Calcium entry via voltage-dependent calcium channels during the AP activates several types of calcium-activated potassium channels. These channels contribute to repolarization of the AP and play a dominant role in the AHP and spike-frequency adaptation during trains of APs. Two different calcium-activated potassium currents underlie the AHP. One current (IAHP) is mediated by apamin-sensitive SK type channels and has relatively rapid kinetics such that the macroscopic current tracks cytosolic calcium, decaying with a time constant of 50–100 ms. The other current (sIAHP) has much slower kinetics lasting for several seconds. In BLA projection neurons, the AHP that follows an AP train is mediated almost entirely by the apamin-insensitive sIAHP (Fig. 1) (Womble and Moises, 1993). In this regard, neurons in the basal nucleus are similar to projection neurons in the lateral amygdala (Faber and Sah, 2002) and pyramidal neurons of CA1 hippocampus (Oh et al., 2000), where blockade of SK channels has little effect on either the AHP or spike-frequency adaptation. Large rises in cytosolic calcium can also be generated by activation of metabotropic receptors coupled to phospholipase C, with subsequent release of calcium from IP3-sensitive ER calcium stores (Power and Sah, 2007). We have shown that mAChR-evoked store release preferentially activates SK calcium-activated potassium channels that are located close to the soma. This store release is also triggered by calcium-induced calcium release after calcium influx during APs and can act to enhance the AHP and reduce neuronal excitability.
In BLA projection neurons, SK channel activation was only observed with focal stimulation of the soma and proximal dendrites. The necessity for proximal stimulation is also seen in other brain regions (Fiorillo and Williams, 2000; Gulledge and Stuart, 2005; Hagenston et al., 2007) and may reflect a proximal distribution of IP3-sensitive calcium stores (Nakamura et al., 2002; Stutzmann et al., 2003; Power and Sah, 2007). Our data show (Figs. 2E, 3B,E) that the time course of activation of SK channels matches the somatic calcium signal but not the calcium signal of the proximal dendrites, suggesting that the outward current activated by calcium released from intracellular stores is primarily caused by activation of SK channels located on or near the soma. SK channels have also been shown recently to be present on dendritic spines in hippocampal and lateral amygdalar neurons (Faber et al., 2005; Ngo-Anh et al., 2005). However, given that IP3-evoked calcium release is primarily restricted to spine-sparse regions of the dendrite (Nakamura et al., 2002; Stutzmann et al., 2003; Power and Sah, 2007), it is unlikely that SK channels on spines would be activated by metabotropic receptor stimulation. Similar results are seen in pyramidal neurons within the prefrontal cortex, where SK channel activation by mGluR stimulation requires somatic invasion of the calcium wave (Hagenston et al., 2007). The relative apamin insensitivity of the AHP evoked by brief trains of APs may be caused by the apparent distribution of SK channels in the soma and spines but not on proximal dendrites. The amplitude of AP-evoked calcium rises is far greater in the dendrites than in the soma (Markram et al., 1995; Sah and Clements, 1999; Power and Sah, 2007), such that SK channels, with an EC50 value for calcium of 300–700 nm (Stocker, 2004), would show limited activation by the modest somatic calcium rise generated by AP trains. SK channels located on spines may be too electrotonically distant or decay too rapidly to have much impact on the somatic membrane potential.
Interestingly, the large rises in cytosolic calcium evoked by cholinergic stimulation do not appear to activate the calcium-activated potassium channels responsible for the sIAHP. There are a number of possible explanations for this result. First, in hippocampal pyramidal neurons, the channels that underlie the sIAHP have been proposed to be present on the dendritic tree (Sah and Bekkers, 1996; Bekkers, 2000). If a similar situation holds in BLA projection neurons, the lack of IP3-mediated calcium release in the distal dendritic tree (Power and Sah, 2007) may explain why they are not activated by an mAChR-evoked calcium response. Second, activation of the sIAHP has been proposed recently to occur through an interaction with a diffusible calcium sensor (Tzingounis et al., 2007). Thus, it is conceivable that this calcium sensor in BLA neurons does not have access to calcium released from internal stores. Finally, given that the sIAHP is suppressed by mAChR activation, it is possible that cholinergic suppression of the sIAHP occurs before store release.
Because of the calcium dependence of the IP3 receptor, calcium store release is augmented when AP-evoked calcium influx occurs coincidently with mAChR stimulation (Fig. 4). In pyramidal neurons in visual cortex, where the AHP is predominantly mediated by the IAHP, IP3-CICR results in an enhancement of the AHP and spike-frequency adaptation (Yamada et al., 2004). In BLA projection neurons, the situation is more complex, because the sIAHP is the predominant current responsible for the AHP and spike-frequency adaptation. Results from the differential blockade of SK channels and the sIAHP demonstrate that a competition between these calcium-activated potassium channels determines the cholinergic action on the AHP and spike-frequency adaptation. In the absence of IP3-CICR, mAChR stimulation suppresses the AHP and reduces spike frequency adaptation. When IP3-CICR is functional, the resultant activation of SK channels counteracts the cholinergic suppression of the AHP and spike-frequency adaptation. This store-dependent calcium rise and SK activation lasts for several seconds, significantly longer than the IAHP in the absence of metabotropic stimulation. Both the IP3-CICR and the inhibitory effects of ACh are transient and are followed by a longer-lasting reduction in spike frequency adaptation, presumably because of the cholinergic suppression of the potassium channels and activation of mixed cation conductances (Washburn and Moises, 1992b; Womble and Moises, 1993; Yajeya et al., 1999). Because store content and its release potential are dependent on previous neuronal activity (Power and Sah, 2005), the calcium-store signal also conveys information about previous neuronal activity (Fig. 3D). Thus, the dependence of SK activation on calcium-store release, and the preferential location of these channels on the soma, close to the site of AP generation, provides a mechanism for coincident activity to modulate intrinsic excitability.
What is the functional role of the metabotropic-mediated hyperpolarization? It has been proposed that activation of calcium-activated potassium channels may be neuroprotective, delaying the onset of epileptogenesis during periods of enhanced neuronal activity (Rainnie et al., 1994). SK channel activation may also act to uncouple large postsynaptic calcium rises from neuronal output, ensuring that calcium-dependent changes necessary for synaptic plasticity are not passed on to downstream targets of BLA projection neurons. Interestingly, learning-associated reductions in the sIAHP are well correlated with learning and memory in the hippocampus and piriform cortex (Saar and Barkai, 2003; Disterhoft and Oh, 2006). Although it is unclear whether a similar reduction of the sIAHP occurs in BLA neurons, noradrenaline levels in the BLA have been shown to increase in response to emotionally arousing stimuli such as footshocks (Galvez et al., 1996). Our data show that a reduction of the sIAHP, through adrenergic stimulation or other mechanisms, enhances the transient inhibitory effects of cholinergic stimulation. This biphasic action of cholinergic stimulation on the AHP and spike-frequency adaptation may serve to tune neuronal output. Excitatory inputs that immediately follow cholinergic stimulation would be inhibited from producing APs, whereas inputs that are predictive of the cholinergic stimulation are unaffected. Excitatory inputs occurring after this brief window of inhibition are more likely to initiate neuronal output. Continuous excitation, which would normally result in a burst of APs that rapidly accommodate, may yield two bursts of APs, one before and one after the onset of cholinergic activity.
This work was supported by grants from the National Health and Medical Research Council of Australia and the Australian Research Council. J.M.P. is a recipient of a Smart State Fellowship from the Queensland State Government. We thank Matt Oh and Rowan Tweedale for comments on this manuscript.
- Correspondence should be addressed to either John Power or Pankaj Sah at the above address. or
- Barros et al., 2005.↵
- Bekkers, 2000.↵
- Ben-Ari et al., 1977.↵
- Bezprozvanny et al., 1991.↵
- Chen and Lang, 2003.↵
- Chen et al., 2000.↵
- Disterhoft and Oh, 2006.↵
- Dutar and Nicoll, 1989.↵
- Faber and Sah, 2002.↵
- Faber and Sah, 2005.
- Faber et al., 2005.↵
- Faber et al., 2006.↵
- Finch et al., 1991.↵
- Fiorillo and Williams, 1998.↵
- Fiorillo and Williams, 2000.↵
- Galvez et al., 1996.↵
- Gu et al., 2005.↵
- Gulledge and Stuart, 2005.↵
- Hagenston et al., 2007.↵
- Huang et al., 1994.↵
- Jaffe and Brown, 1994.↵
- Klein and Yakel, 2006.↵
- Krause and Pedarzani, 2000.↵
- Kroner et al., 2005.↵
- Lang and Pare, 1997.↵
- Levey et al., 1991.↵
- Mak et al., 1999.↵
- Markram et al., 1995.↵
- Mash and Potter, 1986.↵
- McGaugh, 2004.↵
- Morikawa et al., 2003.↵
- Muller et al., 1992.↵
- Nakamura et al., 1999.↵
- Nakamura et al., 2002.↵
- Ngo-Anh et al., 2005.↵
- Nicoll, 1988.↵
- Oh et al., 2000.↵
- Pare and Gaudreau, 1996.↵
- Pare et al., 1995.↵
- Pedarzani and Storm, 1993.↵
- Power and Sah, 2002.↵
- Power and Sah, 2005.↵
- Power and Sah, 2007.↵
- Rainnie et al., 1993.↵
- Rainnie et al., 1994.↵
- Ross et al., 2005.↵
- Saar and Barkai, 2003.↵
- Sah, 1996.↵
- Sah and Bekkers, 1996.↵
- Sah and Clements, 1999.↵
- Sah and Faber, 2002.↵
- Schnee and Brown, 1998.↵
- Stocker, 2004.↵
- Stutzmann et al., 2003.↵
- Taylor and Marshall, 1992.↵
- Tinsley et al., 2004.↵
- Tzingounis et al., 2007.↵
- Washburn and Moises, 1992a.↵
- Washburn and Moises, 1992b.↵
- Watanabe et al., 2006.↵
- Watanabe et al., 1995.↵
- Womble and Moises, 1993.↵
- Womble and Moises, 1994.↵
- Yajeya et al., 1999.↵
- Yamada et al., 2004.↵
- Zhu et al., 2005.↵