α₇-Containing nicotinic acetylcholine receptors on interneurons of the basolateral amygdala and their role in the regulation of the network excitability

Abstract

The basolateral amygdala (BLA) plays a key role in fear-related learning and memory, in the modulation of cognitive functions, and in the overall regulation of emotional behavior. Pathophysiological alterations involving hyperexcitability in this brain region underlie anxiety and other emotional disorders as well as some forms of epilepsy. GABAergic interneurons exert a tight inhibitory control over the BLA network; understanding the mechanisms that regulate their activity is necessary for understanding physiological and disordered BLA functions. The BLA receives dense cholinergic input from the basal forebrain, affecting both normal functions and dysfunctions of the amygdala, but the mechanisms involved in the cholinergic regulation of inhibitory activity in the BLA are unclear. Using whole cell recordings in rat amygdala slices, here we demonstrate that the α₇-containing nicotinic acetylcholine receptors (α₇-nAChRs) are present on somatic or somatodendritic regions of BLA interneurons. These receptors are active in the basal state enhancing GABAergic inhibition, and their further, exogenous activation produces a transient but dramatic increase of spontaneous inhibitory postsynaptic currents in principal BLA neurons. In the absence of AMPA/kainate receptor antagonists, activation of α₇-nAChRs in the BLA network increases both GABAergic and glutamatergic spontaneous currents in BLA principal cells, but the inhibitory currents are enhanced significantly more than the excitatory currents, reducing overall excitability. The anxiolytic effects of nicotine as well as the role of the α₇-nAChRs in seizure activity involving the amygdala and in mental illnesses, such as schizophrenia and Alzheimer's disease, may be better understood in light of the present findings.

the amygdala is a group of nuclei in the temporal lobe that receives information from all sensory modalities, locally processes this information for its emotional significance, and plays a key role in the orchestration of the behavioral response (Pitkanen et al. 1995; Sah et al. 2003). The amygdala is particularly responsive to fear-evoking stimuli, and it appears to be the site where fear-related memories are consolidated and stored (Gale et al. 2004; LeDoux 2003, 2005; Muller et al. 1997; Pape and Pare 2010). Amygdalar dysfunction that involves hyperexcitability and hyperactivity is a key feature of anxiety disorders, including posttraumatic stress disorder (Davis et al. 1994; Etkin and Wager 2007; Shin et al. 2004). Furthermore, the amygdala is very prone to seizure generation, and, along with the hippocampus, it is the epileptic focus in certain types of epilepsy (Aroniadou-Anderjaska et al. 2008; Gloor 1992; Pitkanen et al. 1998). Therefore, knowledge of the mechanisms that regulate the excitability of the amygdala can have significant implications in understanding the pathophysiology of and treatment for anxiety and seizure disorders.

Neuronal excitability in the basolateral nucleus of the amygdala (BLA) is particularly relevant to both anxiety (Davis 1998; Davis et al. 1994; Etkin et al. 2004; Etkin and Wager 2007; LeDoux 2003) and seizure generation (Aroniadou-Anderjaska et al. 2008). GABAergic inhibition plays a primary role in the regulation of the excitability of neuronal networks (e.g., Lang and Pare 1998). Although GABAergic interneurons in the BLA make up only a small proportion (∼20%) of the total neuronal population (McDonald and Mascagni 2002; Sah et al. 2003; Spampanato et al. 2011), they tightly regulate principal cell excitability (Lang and Pare 1998; Spampanato et al. 2011; Woodruff and Sah 2007). A number of neuromodulatory systems participate in the regulation of GABAergic synaptic transmission in the BLA (Aroniadou-Anderjaska et al. 2007; Braga et al. 2003, 2004; Kishimoto et al. 2000; Ohshiro et al. 2011; Rainnie 1999). The cholinergic system is prominently present in the BLA (Ben-Ari et al. 1977; Muller et al. 2011; Power 2004), but its involvement in the regulation of GABAergic synaptic transmission is not well-understood.

The cholinergic projections to the BLA arise primarily from the nucleus basalis magnocellularis (Carlsen et al. 1985; Emson et al. 1979; Nagai et al. 1982; Woolf 1991), a collection of neurons in the substantia innominata of the basal forebrain. Afferents from these neurons synapse on both pyramidal cells and interneurons (Carlsen and Heimer 1986; Muller et al. 2011; Nitecka and Frotscher 1989), targeting nicotinic and/or muscarinic acetylcholine receptors, which are abundantly present in the BLA (Hill et al. 1993; Mash and Potter 1986; Muller et al. 2011; Segal et al. 1978; Swanson et al. 1987; van der Zee and Luiten 1999; Zhu et al. 2005).

Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric and are composed, in different subunit combinations, of α₂–α₁₀ and β₂–β₄ subunits (Alkondon and Albuquerque 2004; Dani 2001; Dani and Bertrand 2007; McGehee and Role 1995). The homomeric α₇ and heteromeric α₄β₂ are the two major subtypes of nAChRs found in the mammalian brain (Albuquerque et al. 2009; Gotti et al. 2009). α₇-nAChRs play an important role in the regulation of neuronal excitability in different brain regions either by presynaptically modulating neurotransmitter release (Barik and Wonnacott 2006; Dickinson et al. 2008; Livingstone et al. 2009; Quarta et al. 2009) or by their position on somatodendritic sites of interneurons and pyramidal cells, where they directly regulate neuronal activity (Alkondon and Albuquerque 2001; Alkondon et al. 1996, 1998, 2000; Arnaiz-Cot et al. 2008; Kalappa et al. 2010; Khiroug et al. 2003; Klein and Yakel 2006). In the BLA, α₇-nAChRs are present on somatodendritic regions of glutamatergic neurons (Klein and Yakel 2006), and they are also involved in presynaptically facilitating glutamate release (Barazangi and Role 2001; Jiang and Role 2008). It is unclear whether α₇-nAChRs are also present on somatodendritic sites of BLA interneurons. There is only one study in the literature addressing this question, where it is reported that in the BLA of neonatal [postnatal day 7 (P7)-10] rats, an increase in the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) by application of acetylcholine or nicotine was not reduced by the specific α₇-nAChR antagonists α-bungarotoxin (α-BgTx) or methyllycaconitine, suggesting that α₇-nAChRs do not play an important role in the regulation of GABAergic activity in the BLA (Zhu et al. 2005). The focus of the present study was to delineate the role of α₇-nAChRs in the regulation of GABAergic activity in the BLA and determine the net effect of α₇-nAChR activation on the excitability of the BLA network.

METHODS

Animals.

Experiments were performed using 25- to 40-day-old, male, Sprague-Dawley rats (Taconic Farms, Derwood, MD). Animals were housed in an environmentally controlled room [20–23°C, 44% humidity, 12:12-h light-dark cycle (350–400 lx), lights on at 6:00 AM] with food (2018 Teklad Global Diet, 18% protein rodent diet; Harlan Laboratories, Indianapolis, IN) and water available ad libitum. Cages were cleaned weekly and had no physical enrichment within the cage (Prager et al. 2011). All animal experiments were conducted following the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council) and were approved by the Institutional Animal Care and Use Committee.

Electrophysiological experiments.

Animals were anesthetized with isoflurane before decapitation. Coronal brain slices (400 μm thick) containing the amygdala (−2.64 to −3.36 from bregma) were cut using a vibratome (Leica VT1200 S; Leica Microsystems, Buffalo Grove, IL) in ice-cold cutting solution consisting of (in mM): 115 sucrose, 70 N-methyl-d-glucamine (NMDG), 1 KCl, 2 CaCl₂, 4 MgCl₂, 1.25 NaH₂PO₄, 30 NaHCO₃, and 25 d-glucose. The slices were transferred to a holding chamber, at room temperature, in a bath solution containing (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 21 NaHCO₃, 2 CaCl₂, 1 MgCl₂, and 11 d-glucose. Recording solution was the same as the holding bath solution. For field potential recordings, the bath/recording solution was same as above except for the concentration of MgCl₂ (1.5 mM) and KCl (3 mM). All solutions were saturated with 95% O₂-5% CO₂ to achieve a pH near 7.4. For whole cell recordings, the slice chamber (0.7-ml capacity) had continuously flowing artificial cerebrospinal fluid (ACSF; ∼8 ml/min) at temperature 32∼33°C. The osmolarity of this external solution was adjusted to 325 mosM with d-glucose. Field potential recordings were obtained in an interface-type chamber, maintained at 32∼33°C, with a flow rate of the ACSF at 1.5 ml/min.

For whole cell recordings, neurons were visualized under infrared light using Nomarski optics of an upright microscope (Axioskop 2; Zeiss, Thornwood, NY) through a ×40 water-immersion objective, equipped with a CCD-100 camera (Dage-MTI, Michigan City, IN). The patch electrodes had resistances of 3.5–4.5 MΩ when filled with the internal solution (in mM): 60 CsCH₃SO₃, 60 KCH₃SO₃, 10 KCl, 10 EGTA, 10 HEPES, 5 Mg-ATP, 0.3 Na₃GTP (pH 7.2), 290 mosM. When sIPSCs and spontaneous excitatory postsynaptic currents (sEPSCs) were recorded simultaneously, the internal chloride concentration was 1 mM, and osmolarity was adjusted with potassium gluconate. We use KCl bridge electrode holders (ALA Scientific Instruments, Farmingdale, NY), which provide stable offset potentials and make the concentration of Cl⁻ in the pipette solution irrelevant (the Ag⁺/AgCl wires are in constant contact with 2 M KCl). Tight-seal (>1 GΩ) whole cell recordings were obtained from the cell body of pyramidal-shaped neurons in the BLA region and from the cell body of interneurons, which were identified on the basis of their electrophysiological properties (Park et al. 2007; Sah et al. 2003). Access resistance (15–24 MΩ) was regularly monitored during recordings, and cells were rejected if the resistance changed by >15% during the experiment. Agonists of α₇-nAChRs were applied either to the bath or by pressure injection. Pressure application was performed with the help of a push-pull experimental arrangement (Pidoplichko and Dani 2005) as used previously (Figueiredo et al. 2011; Williams et al. 2011). A motorizer (Newport, Fountain Valley, CA) was coupled with the approach/withdrawal (push-pull) actuator of a micromanipulator (Burleigh PCS-5000 series; EXFO Photonic Solution, Mississauga, Ontario, Canada). Motorizer movement and duration of application pulses were controlled with a Master-8 digital stimulator (AMPI, Jerusalem, Israel). The pipette was placed ∼50 μm away from the soma of the recorded neuron, and pressure was applied for 70–100 ms via a Picospritzer (General Valve Division, Parker Hannifin, Fairfield, NJ) set between 14 and 30 psi. Ionic currents and action potentials were amplified and filtered (1 kHz) using the Axopatch 200B amplifier (Axon Instruments, Foster City, CA) with a four-pole, low-pass Bessel filter, digitally sampled (up to 2 kHz) using the pClamp 10.2 software (Molecular Devices, Sunnyvale, CA), and further analyzed using the Mini Analysis Program (Synaptosoft, Fort Lee, NJ) and Origin (OriginLab, Northampton, MA). In some experiments, the charge transferred by postsynaptic currents was calculated using the Mini60 software by Synaptosoft.

Field potentials were evoked by stimulation of the external capsule at 0.05 Hz. Recording glass pipettes were filled with ACSF and had a resistance of ∼5 MΩ. Stimulation was applied with a bipolar concentric stimulating electrode made of tungsten (World Precision Instruments, Sarasota, FL). Signals were digitized using the pClamp 10.2 software, analyzed using Clampfit 10.2, and final presentation was prepared using Origin or GraphPad Prism (GraphPad Software, La Jolla, CA).

Drugs used were as follows: bicuculline methiodide, a GABA_A receptor antagonist; atropine sulphate, a muscarinic AChR antagonist; dihydro-β-erythroidine (DHβE), an α₄β₂-nicotinic receptor antagonist; choline chloride and tricholine citrate, α₇-agonists; α-BgTx, an α₇-antagonist; and CNQX, an AMPA/kainate receptor antagonist (all purchased from Sigma-Aldrich, St. Louis, MO). We also used d-AP5, an N-methyl-d-aspartate receptor antagonist, SCH 50911, a GABA_B receptor antagonist, α-conotoxin Au1B, an α₃β₄-nicotinic receptor antagonist, and LY 341495, a metabotropic glutamate group II/III receptor antagonist (all purchased from Tocris Bioscience, Ellisville, MO).

Statistical analysis.

Electrophysiological studies were analyzed using paired Student's t-tests. Results were considered statistically significant when P < 0.05. Data are presented as means ± standard error of the mean. Sample size “n” refers to the number of neurons for the whole cell experiments and the number of slices for the field potential recordings.

RESULTS

Functional α₇-nAChRs are present on BLA interneurons.

Whole cell recordings were obtained from presumed interneurons in the BLA, which were identified on the basis of their small size compared with pyramidal/principal cells, their firing pattern in response to depolarizing current pulses in the current-clamp mode, and the absence of a current activated by hyperpolarizing voltage steps, in the voltage-clamp mode. Depolarizing current injections generated a high-frequency series of fast, nonaccommodating action potentials (Fig. 1B, right), which is typical of a significant subpopulation of BLA interneurons (Rainnie et al. 2006; Sah et al. 2003; Szinyei et al. 2000). Voltage-clamp recordings demonstrated linear change in leakage current and the absence of I_h (Fig. 1B, right), which is a cationic current activated by hyperpolarizing voltage steps that is commonly present in principal neurons of the BLA (Aroniadou-Anderjaska et al. 2012; Park et al. 2007; Womble and Moises 1993).

Fig. 1.

α₇-Containing nicotinic acetylcholine receptors (α₇-nAChRs) are present on basolateral amygdala (BLA) interneurons. Recordings were obtained from electrophysiologically identified interneurons in the BLA, in the presence of α-conotoxin Au1B (1 μM), dihydro-β-erythroidine (DHβE; 10 μM), atropine sulfate (0.5 μM), N-methyl-d-aspartate receptor antagonist d-AP5 (50 μM), AMPA/kainate receptor antagonist CNQX (20 μM), GABA_B receptor antagonist SCH 50911 (10 μM), LY 3414953 (3 μM), and bicuculline (20 μM). A: examples of fast and slow currents evoked by pressure application of 10 mM choline chloride [arrowhead; 100 ms; 30 psi; holding potential (V_h) = −70 mV; internal Cl⁻ concentration = 10 mM]. The insets show the absence of the current activated by hyperpolarization (I_h). B: in the current-clamp (C-clamp) mode, pressure application of 5 mM tricholine citrate (arrowhead) induced brief spiking [top left; the membrane potential (V_m) was held at −60 mV by passing low-amplitude depolarizing current]. The bottom left shows the inward current evoked in the same cell by tricholine citrate (5 mM; arrowhead) in the voltage-clamp (V-clamp) mode [V_h, −70 mV]. The right shows the fast, nonaccommodating spiking of the same neuron in response to depolarizing current injections and the absence of a “sag” in response to hyperpolarizing current injections (top) as well as the linear changes in leakage current (absence of I_h) during 1-s long, 10-mV hyperpolarizing steps, starting from the holding potential of −70 mV (bottom). C: the fast current activated by sequential (40-s interval) pressure application of 10 mM choline chloride (arrowheads; same settings as in A) was blocked by bath application of either 250 nM α-bungarotoxin (α-BgTx; top trace) or 1 μM α-BgTx (bottom trace). Top and bottom traces are from 2 different neurons. Gray bars over the recordings mark the duration of bath application of α-BgTx. D: example of the low sensitivity of choline-evoked, slow currents to α-BgTx. Arrowheads show the time point of pressure application of 10 mM choline chloride. α-BgTx was bath-applied initially at the concentration of 500 nM followed by 1 μM (duration of application, 8 min at each concentration; flow rate, 8 ml/min).

To determine whether α₇-nAChRs are present on BLA interneurons, we pressure-applied tricholine citrate (5 mM, n = 7) or choline chloride (10 mM, n = 13) while recording from interneurons in the presence of α-conotoxin Au1B (1 μM), DHβE (10 μM), atropine sulfate (0.5 μM), d-AP5 (50 μM), CNQX (20 μM), SCH 50911 (10 μM), LY 3414953 (3 μM), and bicuculline (20 μM). The concentrations of the α₇-nAChR agonists that we used have been shown in the hippocampus (Alkondon et al. 2007) or neocortical layer I interneurons (Christophe et al. 2002) to activate α₇-nAChRs selectively; furthermore, the presence of the α₄β₂- and the α₃β₄-nicotinic receptor antagonists in the slice medium can increase confidence that tricholine citrate and choline chloride selectively activated α₇-nAChRs. Out of 20 cells, 12 responded to the “puff application” of the α₇-nAChR agonist with a fast-inactivating inward current of an amplitude 83.1 ± 8.4 pA (Fig. 1A, left trace). The remaining 8 cells responded with a slow current of an amplitude 236 ± 52.1 pA (Fig. 1A, right trace). The fast or slow kinetics of the evoked currents had no relation to whether tricholine citrate or choline chloride was applied. In the current-clamp mode, puff application of the α₇-nAChR agonist elicited a brief train of action potentials (Fig. 1B, left, top trace). The fast-inactivating currents were blocked equally effectively by either 250 nM (n = 4; Fig. 1C, top trace) or 1 μM (n = 5; Fig. 1C, bottom trace) α-BgTx within 2–4 min of bath application of the toxin; the mean reduction was 94.5 ± 2.0% (n = 9). The slow currents were only partially reduced by 250 nM, 500 nM, or 1 μM α-BgTx. The blockade (36.5 ± 4.8%, n = 6) was slow (required at least 8 min of perfusion) and appeared to be more effective at the high concentration of the toxin (1 μM; Fig. 1D).

Glutamatergic excitation of interneurons following α₇-nAChR activation.

The experiments described above indicate that α₇-nAChRs are present on somatic or somatodendritic regions of BLA interneurons, and their activation depolarizes these cells. Since α₇-nAChRs are also present on principal BLA neurons (Klein and Yakel 2006) as well as on glutamatergic terminals (Barazangi and Role 2001; Jiang and Role 2008), we investigated whether α₇-nAChR activation in the BLA network, in addition to depolarizing interneurons directly, also depolarizes interneurons by increasing glutamatergic activity. Therefore, we conducted similar experiments as those shown in Fig. 1 except that CNQX was not included in the slice medium. Under these conditions, pressure application of 5 mM tricholine citrate induced long-lasting, high-frequency firing in the current-clamp mode and a transient inward current accompanied by sEPSCs in the voltage-clamp mode (n = 7; Fig. 2A). Similarly, when choline chloride (2.5 mM) was applied to the bath, it also induced repetitive, high-frequency firing of BLA interneurons (n = 4; Fig. 2B). Thus α₇-nAChR activation in the BLA increases the activity of interneurons not only via direct depolarization, but also indirectly due to an enhancement of glutamatergic activity.

Fig. 2.

Indirect excitation of interneurons by activation of α₇-nAChRs. Recordings are from electrophysiologically identified interneurons in the presence of α-conotoxin Au1B (1 μM), DHβE (10 μM), atropine sulfate (0.5 μM), d-AP5 (50 μM), SCH 50911 (10 μM), LY 3414953 (3 μM), and bicuculline (20 μM). A: pressure application of tricholine citrate (5 mM; arrowhead) induced high-frequency firing in the current-clamp mode (top left) and a transient inward current along with high-frequency spontaneous excitatory postsynaptic currents (sEPSCs) in the voltage-clamp mode (bottom left; V_h, −70 mV). The electrophysiological characteristics of this neuron are shown on the right. Responses to depolarizing or hyperpolarizing current injections (top right) and hyperpolarizing voltage steps (bottom right) are consistent with the properties of interneurons. B: in current-clamp mode, bath application of choline chloride (2.5 mM) induced high-frequency spiking.

Activation of α₇-nAChRs enhances sIPSCs.

The neurons we recorded from in the experiments described above were presumed to be GABAergic interneurons. Therefore, the data presented would predict that activation of α₇-nAChRs will enhance GABAergic inhibition in the BLA. To determine whether α₇-nAChR activation increases GABA_A receptor-mediated sIPSCs, we bath-applied choline chloride while recording from principal neurons (n = 12) in the presence of α-conotoxin Au1B (1 μM), DHβE (10 μM), atropine sulfate (0.5 μM), d-AP5 (50 μM), CNQX (20 μM), SCH 50911 (10 μM), and LY 3414953 (3 μM). Neurons were identified as principal cells based on their size and pyramidal-like shape as well as on their firing patterns in response to depolarizing current pulses and the presence of I_h (Aroniadou-Anderjaska et al. 2012; Park et al. 2007; Sah et al. 2003). Choline chloride (5 mM) was bath-applied at a flow rate of 8 ml/min. The effect was immediate and consisted of the appearance of a barrage of sIPSCs (Fig. 3). The frequency of sIPSCs was increased by choline from 22 ± 1 to 50 ± 3 Hz (n = 12; P < 0.001); the measurements of the sIPSC frequency in the presence of choline were made for a 20-s time window within a time period of 30 s after the initiation of the effect. The effect subsided within 40 s to 2 min despite the continual presence of choline, suggesting desensitization of the receptors. Reapplication of choline after only partial washout of the initially applied choline had virtually no effect, probably due to the lasting desensitization of the receptors while the exogenous choline was still present. However, after washing out choline for at least 8 min, reapplication of choline could evoke a similar effect, suggesting that desensitization was not lasting after removal of the 5 mM exogenous agonist. Higher concentrations of choline (we tried 10 and 20 mM) produced a similar increase in sIPSC amplitude and frequency, but longer washout times were required to be able to reproduce the effect on reapplication of choline.

Fig. 3.

Activation of α₇-nAChRs enhances spontaneous GABA_A receptor-mediated inhibitory postsynaptic currents (sIPSCs). Recordings are from BLA principal cells in the presence of α-conotoxin Au1B (1 μM), DHβE (10 μM), atropine sulfate (0.5 μM), d-AP5 (50 μM), CNQX (20 μM), SCH 50911 (10 μM), and LY 3414953 (3 μM) at V_h +30 mV and internal chloride 10 mM. A: bath application of choline chloride (5 mM) induced a transient outward cationic current and increased the frequency and amplitude of sIPSCs. The sIPSCs were blocked by 20 μM bicuculline, indicating that they were mediated by GABA_A receptors. B: amplitude-frequency histogram of the effect of choline on sIPSCs (n = 12; ***P < 0.001). C: group data (mean ± SE) of the change in the frequency of sIPSCs by bath application of choline chloride (n = 12; ***P < 0.001). D: pretreatment of the slices with α-BgTx reduced significantly the effects of choline. Traces show an example of the response of a principal neuron to bath application of choline chloride (5 mM) and the response of the same neuron when choline chloride was applied in the presence of α-BgTx (1 μM). The group data are shown in the bar graphs below (***P < 0.001, **P < 0.01; n = 9).

In some experiments, we washed out choline for 8 min and then applied α-BgTx (1 μM) for another 4 min. Subsequent application of 5 mM choline chloride (in the presence of α-BgTx) increased the frequency of sIPSCs from 18 ± 3 to 37 ± 4 Hz (n = 9; P < 0.01; Fig. 3D). Thus, even in the presence of α-BgTx, choline increased sIPSCs significantly, probably because of the activation of interneurons that respond to choline by generating a slow current with low sensitivity to α-BgTx (Fig. 1D). However, the increase in the frequency of sIPSCs in the presence of α-BgTx was significantly smaller than the increase in the absence of the α₇-nAChR antagonist (P < 0.001; Fig. 3D), probably reflecting the blockade of the α-BgTx-sensitive α₇-nAChRs on interneurons that generate fast-inactivating currents in response to choline (Fig. 1C).

The net effect of α₇-nAChR activation in the BLA.

Since activation of α₇-nAChRs in the BLA can increase both glutamatergic and GABAergic activity, an important question that arises is whether the overall, net effect of α₇-nAChR activation is enhancement or suppression of excitatory activity in the BLA. To answer this question, we recorded both sIPSCs and sEPSCs simultaneously from principal neurons in the presence of α-conotoxin Au1B (1 μM), DHβE (10 μM), atropine sulfate (0.5 μM), d-AP5 (50 μM), SCH 50911 (10 μM), and LY 3414953 (3 μM) at a holding potential of −58 mV and internal chloride concentration of 1 mM. Bath application of choline chloride (5 mM) increased both sIPSCs and sEPSCs (Fig. 4). Out of nine cells, in three cells there was only a small increase in sEPSCs, whereas sIPSCs were dramatically increased (as in the example shown in Fig. 4A), in five cells both sEPSCs and sIPSCs were increased, but the increase in sIPSCs was clearly more prominent (as in the example shown in Fig. 4B), whereas one cell showed dramatic increase in both sIPSCs and sEPSCs. To quantify these effects, we calculated the total charge transferred. The charge, in picocoulombs, was calculated as the area delimited by the inhibitory or excitatory current and the baseline. Current areas were analyzed for a time period of 5 s. On average, the charge transferred by sIPSCs was increased from 1.3 ± 0.3 pC in control medium to 97 ± 24 pC in the presence of choline (n = 9; P < 0.01; Fig. 4C, left), whereas the charge transferred by sEPSCs was increased from 1.5 ± 0.3 to 20 ± 4 pC (n = 9; P < 0.01; Fig. 4C, right) during the initial phase of bath application of choline chloride (during a 5-s period after the initiation of the effect). The charge transferred by sIPSCs was significantly larger than the charge transferred by sEPSCs (n = 9; P < 0.01), suggesting that the net effect of α₇-nAChR activation is inhibitory. As in the case where only sIPSCs were recorded (Fig. 3), the effects of choline subsided in less than 2 min despite the continual presence of the agonist. Lower concentrations of bath-applied choline chloride (1 mM, n = 2, and 0.5 mM, n = 3) had qualitatively similar effects (Fig. 4D).

Fig. 4.

Effects of α₇-nAChR activation on simultaneously recorded sIPSCs and sEPSCs. Recordings were obtained from principal neurons in the presence of α-conotoxin Au1B (1 μM), DHβE (10 μM), atropine sulfate (0.5 μM), d-AP5 (50 μM), SCH 50911 (10 μM), and LY 3414953 (3 μM) at a holding potential of −58 mV and internal chloride concentration of 1 mM. A: bath application of choline chloride (5 mM) induced a transient increase in the frequency and amplitude of both sIPSCs (outward currents) and sEPSCs (inward currents), but the increase in sIPSCs was more pronounced. B: another example of the effects of choline chloride (5 mM) on sIPSCs and sEPSCs. The increase in sEPSCs is more apparent in this cell compared with the cell in A. C: group data (mean ± SE) showing the effects of 5 mM choline chloride on sIPSCs (left) and sEPSCs (right). Application of choline significantly increased the mean charge transferred by sIPSCs and sEPSCs (n = 9; **P < 0.01) measured during the 5-s period after the initiation of the effect. During this 5-s period, the charge transferred by sIPSCs was significantly larger than the charge transferred by sEPSCs (n = 9; **P < 0.01). D: lower concentrations of choline produced a similar effect; an example is shown where 500 μM choline chloride was bath-applied.

Since α₇-nAChR activation enhances spontaneous GABAergic activity to a greater extent than glutamatergic activity, this effect should be reflected in the population responses. An enhanced “tonic” inhibition (sIPSCs) in the BLA network can be expected to reduce overall excitability. Field potentials in the BLA are reduced in amplitude by the GABA_A agonist muscimol and are enhanced by bicuculline (E.M. Prager and V. Aroniadou-Anderjaska, unpublished observations), which implies significant presence of synchronized spiking activity (somatic and/or dendritic) in the generation of the field responses. Since choline enhances GABAergic inhibition, the amplitude of evoked population responses should be reduced in the presence of choline. To test this prediction, we bath-applied choline chloride while recording field potentials in the BLA, evoked by stimulation of the external capsule. In these experiments, slices were placed in an interface chamber, which has a much slower flow rate than in the whole cell recording experiments; the gradual exposure of the slice to low and slowly rising concentrations of choline chloride could desensitize the receptors, rendering the effect undetectable. Therefore, we used a high concentration of choline chloride (20 mM) to detect the effect. Choline chloride reversibly decreased the amplitude of the field response from 0.49 ± 0.02 to 0.34 ± 0.02 mV (n = 8; P < 0.001; Fig. 5). In percentages, choline reduced the evoked field potential to 71.87 ± 4.11% of the control amplitude (P < 0.001; Fig. 5C). These results are consistent with the view that activation of α₇-nAChRs reduces the excitability of the BLA network.

Fig. 5.

Activation of α₇-nAChRs reduces evoked field potentials in the BLA. A: an example of field potentials evoked in the BLA by stimulation of the external capsule before, during, and after washout of choline chloride (20 mM). Each trace is an average of 10 sweeps. B: same field potentials as in A, in control medium and in the presence of choline, superimposed for a clearer view of the effect of choline. C: group data from 8 slices showing the amplitude of the field responses as percentages of the control responses before, during, and after bath application of choline (n = 8; ***P < 0.001).

α₇-nAChRs are active in the basal state contributing to background inhibition.

Considering that α₇-nAChRs have low affinity for their endogenous agonists, acetylcholine and choline, and desensitize rapidly (Albuquerque et al. 2009), it is important to ask whether these receptors can be activated by ambient concentrations of acetylcholine or choline, thereby contributing to the background levels of inhibitory and/or excitatory activity. To answer this question, first we examined the effects of bath-applied α-BgTx (1 μM) on sIPSCs recorded from principal BLA neurons. The frequency of sIPSCs was reduced by bath application of α-BgTx from 43 ± 9 Hz in control medium to 22 ± 8 Hz in α-BgTx (n = 4; P < 0.01; Fig. 6, A and B). Next, we tested the effects of α-BgTx on simultaneously recorded sIPSCs and sEPSCs from principal neurons. α-BgTx (1 μM) significantly decreased the frequency of sIPSCs from 24 ± 3 to 12 ± 3 Hz (n = 4; P = 0.0316), whereas the frequency of sEPSCs was reduced from 22 ± 4 Hz in control medium to 15 ± 3 Hz in the presence of α-BgTx (n = 4; P = 0.141). These results suggest that basal activation of α₇-nAChRs on interneurons (the postsynaptic α₇-nAChRs demonstrated in the present study and/or presynaptic α₇-nAChRs on GABAergic terminals) contributes significantly to spontaneous inhibitory activity.

Fig. 6.

Blockade of α₇-nAChRs in the basal state decreases the frequency of sIPSCs. A: an example from a principal cell displaying high inhibitory synaptic activity in control medium and the effect of α-BgTx (1 μM) on sIPSCs (V_h = +30 mV); the reduction of the sIPSCs by α-BgTx was irreversible. The slice medium contains α-conotoxin Au1B (1 μM), DHβE (10 μM), atropine sulfate (0.5 μM), CNQX (20 μM), d-AP5 (50 μM), SCH 50911 (10 μM), and LY 3414953 (3 μM). B: group data showing the effect of α-BgTx on the frequency of sIPSCs (n = 4; **P < 0.01). C: an example from another principal cell where sIPSCs and sEPSCs were recorded simultaneously (V_h = −58 mV, internal chloride concentration is 1 mM). The reduction of the sIPSCs by α-BgTx (1 μM) was more pronounced than the reduction of sEPSCs. The slice medium contains α-conotoxin Au1B (1 μM), DHβE (10 μM), atropine sulfate (0.5 μM), d-AP5 (50 μM), SCH 50911 (10 μM), and LY 3414953 (3 μM). D: group data showing the effect of α-BgTx on the frequency of sIPSCs (n = 4; *P < 0.05) and sEPSCs (n = 4; P > 0.05).

DISCUSSION

This study demonstrated the presence of functional α₇-nAChRs on somatic or somatodendritic regions of electrophysiologically identified interneurons in the BLA. Activation of α₇-nAChRs by choline directly depolarized interneurons and elicited action potentials. In addition, α₇-nAChR activation increased the frequency of sEPSCs recorded from interneurons, probably due to α₇-nAChR-mediated depolarization of principal, glutamatergic neurons and/or presynaptic facilitation of glutamate release. The increase in interneuronal activity by α₇-nAChR activation produced a dramatic increase in the frequency and amplitude of GABA_A receptor-mediated sIPSCs recorded from BLA principal cells. Simultaneous recordings of sIPSCs and sEPSCs from principal neurons revealed that activation of α₇-nAChRs by choline increases both types of currents, but the increase of the inhibitory currents is larger than the increase of the excitatory currents. This observation, along with the reduction of the population field response in the presence of choline, suggests that the net effect of α₇-nAChR activation in the BLA network is suppression of excitability. This function of α₇-nAChRs appears to be in effect even in the basal state, as blockade of these receptors decreased the frequency of sIPSCs.

The α₇-subunit of the nicotinic receptors is expressed in the BLA (Zhu et al. 2005), and functional α₇-nAChRs are present on somatodendritic regions of principal BLA neurons (Klein and Yakel 2006). However, it was unknown whether α₇-nAChRs are also present on somatodendritic regions of BLA interneurons. This is the first study investigating the responses of BLA interneurons to the activation of α₇-nAChRs by a specific agonist. We found that 60% of the recorded interneurons responded with a fast-inactivating current, which was blocked by α-BgTx; these characteristics are typical of the currents mediated by homomeric α₇-nAChRs (Albuquerque et al. 2009; Khiroug et al. 2002). However, the remaining interneurons displayed a slow current, which was only partly sensitive to α-BgTx. Since this current was activated by choline in the presence of antagonists of all cholinergic receptors except for the α₇-nAChRs, it was probably mediated by α₇-nAChRs. The slow kinetics and the partial resistance to α-BgTx may suggest that the α₇-nAChRs mediating this current are not homomeric. A different composition and stoichiometry of these α₇-containing receptors may be responsible for the different kinetics and pharmacology of the currents they mediate. For example, the α₇β₂-subunit combination has different pharmacological properties and produces currents with slower kinetics compared with the homomeric α₇-nAChRs (Khiroug et al. 2002). The presence of heteromeric α₇-containing nicotinic receptors (α₇β₂-subunit combination) has been demonstrated in rat basal forebrain cholinergic neurons (Liu et al. 2009); it is possible, therefore, that such heteromeric α₇-nAChRs exist also in other brain regions, including the BLA.

In regard to the role that α₇-nAChRs play in the regulation of GABAergic activity in the rat BLA, there is only one previous study showing that α₇-nAChR antagonists did not reduce the acetylcholine-induced increase in the frequency of sIPSCs recorded from BLA principal cells; therefore, it was suggested that α₇-nAChRs may not participate in the regulation of inhibitory activity in the BLA (Zhu et al. 2005). One factor that may have contributed to the disparity between our results and those of Zhu et al. (2005) could be the age of the animals used, as the latter study was performed in neonatal rats (P7–10), and, at least in hippocampal interneurons, α₇-nAChR-mediated currents increase with age (Alkondon et al. 2007). Another possibility, which the authors (Zhu et al. 2005) have considered, is that the increase in sIPSCs by acetylcholine [the agonist used in Zhu et al. (2005)] was not reduced by subsequent application of specific α₇-nAChR antagonists because the α₇-nAChRs were already desensitized; the increase in sIPSCs was sustained only by non-α₇-nicotinic receptor subtypes that are slow to desensitize. This explanation for the divergent conclusions regarding the importance of α₇-nAChRs in the regulation of GABAergic activity in the BLA is consistent with the present results where the effect of the specific α₇-nAChR agonist, choline, on the sIPSCs did not last for more than 1–2 min, which probably reflects the fast desensitization of the α₇-nAChRs.

What are the functional implications of the involvement of the α₇-nAChRs in the regulation of GABAergic activity in the BLA? Like the α₇-nAChRs on hippocampal interneurons (Alkondon et al. 1999), the α₇-nAChRs in the BLA appear to be active in the basal state, preferentially increasing inhibitory activity. Therefore, even without significant activation of the cholinergic inputs to the amygdala above the basal level, these receptors may participate in the regulation of normal amygdala functions, primarily by limiting excitation. It should be considered, however, that since α₇-nAChRs are present on both inhibitory and excitatory BLA neurons, their in vivo activation in the functioning BLA may promote either excitatory or inhibitory activity depending on a number of factors such as: 1) the concentration of acetylcholine at the vicinity of α₇-nAChRs and the temporal pattern of its increase, which may differentially affect the desensitization of α₇-nAChRs on principal cells vs. interneurons; 2) the role of presynaptic α₇-nAChRs on glutamatergic terminals (Barazangi and Role 2001; Jiang and Role 2008); and 3) possible effects of other concomitantly acting neurotransmitters and neuromodulators that could potentially alter the neurons' response to α₇-nAChR stimulation. Our results show only that inhibition is favored over excitation when α₇-nAChRs are uniformly activated throughout the BLA network.

Relatively uniform activation of α₇-nAChRs in the BLA can be expected during cigarette smoking, and our results suggest that these receptors may, in part, mediate the anxiolytic effects of nicotine. On an acute basis, nicotine is known to have anxiolytic (Bencan and Levin 2008; Cheeta et al. 2001; Cohen et al. 2009; Kassel and Unrod 2000; Szyndler et al. 2001) and antidepressant effects (Hawkins 1997; Vazquez-Palacios et al. 2004). The amygdala, and the BLA in particular, plays a central role in anxiety and depression (Davis 1998; Davis et al. 1994; Drevets 1999; Etkin et al. 2004; Hamilton et al. 2008; LeDoux 2003; Mitra et al. 2009). Our results suggest that the α₇-nAChR-mediated increase in GABAergic activity in the BLA may be one of the mechanisms by which nicotine suppresses BLA excitability, thereby reducing anxiety and alleviating depression. This view receives support by the finding that systemic administration of an α₇-nAChR agonist, in mice, has anxiolytic effects (Feuerbach et al. 2009). Because of the pronounced but only transient increase of inhibitory activity on activation of α₇-nAChRs, followed by desensitization of the receptor, the anxiolytic effect of nicotine may be stronger when α₇-nAChRs are stimulated in an intermittent fashion, as it probably occurs during cigarette smoking. Because of their low affinity for nicotine (Fenster et al. 1997), the α₇-nAChRs may be transiently activated only during the peak levels of nicotine, whereas they recover from desensitization during the troughs.

Considering that the amygdala is a seizure-prone structure with an important role in certain forms of epilepsy (Aroniadou-Anderjaska et al. 2008), the question arises as to whether α₇-nAChRs, by regulating both GABAergic and glutamatergic activity in the BLA, play a significant role in seizure generation and/or suppression. An involvement of the α₇-nAChR in epilepsy is suggested by the association of juvenile myoclonic epilepsy with a mutation in the gene coding for the α₇-subunit (Elmslie et al. 1997). In addition, systemic administration of an α₇-nAChR agonist in mice showed anticonvulsant potential in the audiogenic seizure paradigm (Feuerbach et al. 2009), which is known to involve the amygdala (Feng and Faingold 2002; Hirsch et al. 1997). This finding along with the present data showing a preferential increase of inhibitory activity in the BLA by α₇-nAChR activation suggest that in the amygdala these receptors may contribute to suppression of seizures.

The α₇-nAChRs are known to be important in learning and memory (Boess et al. 2007; Hellier et al. 2012; Levin 2012; Robinson et al. 2011; Van Kampen et al. 2004), including memory involving the amygdala (Addy et al. 2003). In the BLA of Alzheimer's patients, there is accumulation of β-amyloid protein in GABAergic and glutamatergic neurons (Espana et al. 2010), which interacts and forms a protein complex with the α₇-nAChRs (Dziewczapolski et al. 2009; Parri et al. 2011); the resulting inactivation of α₇-nAChRs is considered to be important in the pathogenesis of Alzheimer's disease (Parri et al. 2011). Alzheimer's patients display impaired fear conditioning (Hamann et al. 2002; Hoefer et al. 2008), a form of fear-related memory, as well as increased fear and anxiety (Espana et al. 2010; Ferretti et al. 2001); these symptoms may be related to the inactivation of α₇-nAChRs by β-amyloid protein, producing hyperexcitability in the BLA.

The involvement of α₇-nAChRs in the regulation of GABAergic inhibition in the BLA may also have implications in the pathophysiology of schizophrenia. The expression and function of α₇-nAChRs is reduced in schizophrenia (Adler et al. 1998; Guan et al. 1999; Martin et al. 2004; Olincy and Stevens 2007). The dysfunction of the amygdala in schizophrenic patients (Aleman and Kahn 2005; Benes 2010; Lawrie et al. 2003) and, in particular, the abnormally high activation of the amygdala during processing of stimuli that should not evoke fear (Hall et al. 2008) may imply dysregulation of inhibitory activity in the BLA, which could be, in part, due to abnormalities in α₇-nAChR function.

In conclusion, we demonstrated that α₇-nAChRs are expressed on somatic and/or dendritic regions of GABAergic interneurons in the BLA and that activation of these receptors increases GABA_A receptor-mediated sIPSCs, even in the basal state. In addition, we showed that although activation of α₇-nAChRs increases both sIPSCs and sEPSCs, the net effect is a preferential enhancement of inhibition. The presence of functional α₇-nAChRs on GABAergic interneurons in the BLA may suggest a site for therapeutic treatments of diseases associated with amygdalar hyperactivity.

GRANTS

This work was supported by the CounterACT Program, National Institutes of Health, Office of the Director and the National Institute of Neurological Disorders and Stroke (Grant 5U01-NS-058162-07), and the Defense Threat Reduction Agency-Joint Science and Technology Office, Medical S&T Division (Grants CBM.NEURO.01.10.US.18 and CBM.NEURO.01.10.US.15).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

V.A.-A. and M.F.M.B. acquired funding for the research; V.I.P., E.M.P., V.A.-A., and M.F.M.B. conception and design of research; V.I.P. and E.M.P. performed experiments; V.I.P. and E.M.P. analyzed data; V.I.P., E.M.P., V.A.-A., and M.F.M.B. interpreted results of experiments; V.I.P. and E.M.P. prepared figures; E.M.P. drafted manuscript; V.A.-A. and M.F.M.B. edited and revised manuscript; V.I.P., E.M.P., V.A.-A., and M.F.M.B. approved final version of manuscript.