ASIC1a activation enhances inhibition in the basolateral amygdala and reduces anxiety

Abstract

The discovery that even small changes in extracellular acidity can alter the excitability of neuronal networks via activation of acid-sensing ion channels (ASICs) could have therapeutic application in a host of neurological and psychiatric illnesses. Recent evidence suggests that activation of ASIC1a, a subtype of ASICs that is widely distributed in the brain, is necessary for the expression of fear and anxiety. Antagonists of ASIC1a, therefore, have been proposed as a potential treatment for anxiety. The basolateral amygdala (BLA) is central to fear generation, and anxiety disorders are characterized by BLA hyperexcitability. To better understand the role of ASIC1a in anxiety, we attempted to provide a direct assessment of whether ASIC1a activation increases BLA excitability. In rat BLA slices, activation of ASIC1a by low pH or ammonium elicited inward currents in both interneurons and principal neurons, and increased spontaneous IPSCs recorded from principal cells significantly more than spontaneous EPSCs. Epileptiform activity induced by high potassium and low magnesium was suppressed by ammonium. Antagonism of ASIC1a decreased spontaneous IPSCs more than EPSCs, and increased the excitability of the BLA network, as reflected by the pronounced increase of evoked field potentials, suggesting that ASIC1a channels are active in the basal state. In vivo activation or blockade of ASIC1a in the BLA suppressed or increased, respectively, anxiety-like behavior. Thus, in the rat BLA, ASIC1a has an inhibitory and anxiolytic function. The discovery of positive ASIC1a modulators may hold promise for the treatment of anxiety disorders.

Keywords: ASIC1a; GABAergic inhibition; acid-sensing ion channels; anxiety; basolateral amygdala; epileptiform activity.

Figure 1.

ASIC1a channels are present on BLA interneurons. a, Typical linear currents (I_h is absent) recorded from presumed interneurons in response to 1 s long hyperpolarizing voltage steps (10 mV increments from the holding potential of −70 mV) in voltage-clamp (V-clamp) mode (left), and an example of fast, nonaccommodating spiking of interneurons in response to 500 ms long current injections in the current-clamp (C-clamp) mode (right). b, Brief (200 ms) pressure application of acidified solution (left) or 40 mm ammonium (right) induced inward currents in interneurons, which were increased by lowering the bath temperature. c, Currents evoked by acidified solution or 40 mm ammonium were blocked by bath-applied flurbiprofen (2 mm). d, In the current-clamp mode, bath application of 5 mm ammonium-induced high-frequency firing. In b and c, V_h is −70 mV. In b, c, and d, recordings are in the presence of CNQX (10 μm), d-AP5 (50 μm), bicuculline (20 μm), and SCH50911 (10 μm).

Figure 2.

ASIC1a channels are present on BLA principal neurons. a, Currents recorded from principal neurons in response to hyperpolarizing voltage steps in voltage-clamp (V-clamp) mode (left; notice the presence of I_h), and an example of accommodating firing in response to current injection in the current-clamp (C-clamp) mode (right). b, Pressure application (200 ms) of acidified solution (left) or 40 mm ammonium (right) induced inward currents in principal cells, which were increased by lowering the bath temperature. c, Currents evoked in principal cells by acidified solution or 40 mm ammonium were blocked by bath-applied flurbiprofen (2 mm). d, In the current-clamp mode, bath application of 5 mm ammonium induced bursts of action potentials. In b and c, V_h is −70 mV. In b, c, and d, recordings are in the presence of CNQX (10 μm), d-AP5 (50 μm), bicuculline (20 μm), and SCH50911 (10 μm).

Figure 3.

Activation of ASIC1a increases spontaneous inhibitory activity. Recordings were obtained from BLA principal cells in the presence of CNQX (10 μm), d-AP5 (50 μm), and SCH50911 (10 μm), at V_h = +30 mV. a, sIPSCs before, during, and after bath application of 5 mm ammonium. b, Amplitude-frequency histogram of sIPSCs before and after bath application of 5 mm ammonium (n = 11 neurons from 4 rats); bin width is 5 pA. c, Group data of the frequency of sIPSCs in control medium, in 5 mm ammonium, and after washing out of ammonium (n = 11); ***p < 0.001 when compared with the control. d, sIPSCs before, during, and after perfusion of the slices with acidified ACSF. e, Amplitude-frequency histogram of sIPSCs in control medium and in pH 6.65 (n = 8 neurons from 3 rats); bin width is 5 pA. f, Group data of the frequency of sIPSCs in control medium, in low pH, and after return to control medium (n = 8); ***p < 0.001 when compared with the control.

Figure 4.

Antagonism of ASIC1a reduces spontaneous inhibitory activity. Recordings were obtained from BLA principal cells in the presence of CNQX (10 μm), d-AP5 (50 μm), and SCH50911 (10 μm), at V_h = +30 mV. a, sIPSCs in control medium, in the presence of bath-applied flurbiprofen (2 mm), and after a 10 min wash. Flurbiprofen suppressed sIPSCs, with no significant effect on the amplitude of GABA_A-mediated currents evoked by pressure-applied GABA (400 μm GABA, 200 ms duration of pressure application). The bottom traces show, in an expanded view, the last 5 s of the upper traces. b, Amplitude-frequency histograms for sIPSCs in control medium and in the presence of 2 mm flurbiprofen, for the cell shown in a (bin size, 10 pA). c, Group data of the frequency of sIPSCs in control medium and in the presence of 2 mm flurbiprofen (n = 7 neurons from 4 rats, ***p < 0.001 when compared with the control). d, sIPSCs before, during, and after bath-applied PcTx1 venom (1:1000 dilution of the 100 μl lyophilized, milked venom). PcTx1 suppressed sIPSCs, with no significant effect on the amplitude of GABA_A-mediated currents evoked by pressure-applied GABA. The bottom traces show, in an expanded view, the last 5 s of the upper traces. e, Amplitude-frequency histograms for sIPSCs in control medium and in the presence of PcTx1, for the cell shown in d (bin size, 10 pA). f, Group data of the frequency of sIPSCs in control medium and in the presence of PcTx1 (n = 4 neurons from 3 rats; **p < 0.01 when compared with the control).

Figure 5.

Activation of ASIC1a increases the excitatory drive of interneurons. Recordings are from interneurons at V_h − 58 mV and in the presence of d-AP5 (50 μm) and SCH50911 (10 μm). a, Lowering the pH of the bath increased the frequency of sEPSCs. The bottom current traces in a are from the same cell as in the top trace, at an expanded view. Bath-applied CNQX (10 μm) blocked the recorded currents. b, Currents evoked by pressure application (200 ms) of acidified ACSF in the absence of CNQX, displayed “riding” EPSCs. c, Pressure application of ammonium (40 mm, 500 ms duration) increased the frequency of sEPSCs. d, Bath application of flurbiprofen (1 mm) decreased the frequency of sEPSCs.

Figure 6.

The net effect of ASIC1a activation is suppression of BLA excitability. a, b, Simultaneous recordings of sIPSCs (outward currents) and sEPSCs (inward currents) were obtained from principal cells at V_h − 58 mV, and in the presence of d-AP5 (50 μm) and SCH50911 (10 μm). Bath application of 5 mm ammonium (a) or acidified solution (b) increased the charge transferred by sIPSCs and sEPSCs; the increase in the charge transferred by sIPSCs was significantly greater than the increase in charge transferred by sEPSCs. Example traces are shown in the top of a and b, and group data are shown in the bar graphs; n = 21 neurons from seven rats in a, and n = 9 neurons from four rats in b; ***p < 0.001 for the comparisons between control and ammonium, or control and pH 6.6; **p < 0.01 for the comparisons between percentage increase in charge transferred by sIPSCs versus percentage increase in charge transferred by sEPSCs. c, Field potentials evoked in the BLA by single-pulse stimulation of the external capsule (top traces), and spontaneous field activity recorded in gap-free mode (bottom traces). Recordings are in medium containing 7 mm K⁺ and zero Mg²⁺, which induced epileptiform activity. Bath application of 8 mm ammonium reduced the evoked field potentials and blocked epileptiform discharges. Each of the three field potentials shown in the top is an average of 10 sweeps; the stimulus artifacts have been truncated for clarity. The equidistant vertical lines in the traces of spontaneous activity are stimulus artifacts, as evoked field potentials were sampled during gap-free recordings, by stimulation applied every 20 s.

Figure 7.

The net effect of ASIC1a antagonism is reduction of inhibition and increased excitability. a, Simultaneous recordings of sIPSCs (outward currents) and sEPSCs (inward currents) were obtained from principal cells at V_h − 58 mV, and in the presence of d-AP5 (50 μm) and SCH50911 (10 μm). Bath application of 2 mm flurbiprofen decreased the frequency of sIPSCs to a greater extent than that of sEPSCs. An example is shown in the top, and group data in the bar graphs (n = 9 neurons from 5 rats, ***p < 0.001 for the comparisons between control and flurbiprofen, and percentage increase in charge transferred by sIPSCs vs percentage increase in charge transferred by sEPSCs). b, Field potentials evoked in the BLA by stimulation of the external capsule. Bath application of 2 mm flurbiprofen reversibly increased the amplitude of the evoked responses. Each trace is an average of 10 sweeps.

Figure 8.

In vivo activation of ASIC1a in the BLA suppresses anxiety-like behavior, while antagonism of ASIC1a increases anxiety. a, Panoramic photomicrograph of a coronal, Nissl-stained brain section, showing the drug injection site. Methylene blue was injected before histological analysis. b, Schematic representation of coronal sections from rat brain (from Paxinos and Watson, 2005) showing the injection sites. Coordinates are with respect to bregma. The “dots” (small filled circles) represent the locations of the tips of the guide cannulae, as revealed by histological analysis; their number is fewer than the total number of rats used because of several overlaps. c, d, In the open field test, the rats spent significantly more time in the center, after microinjection of ammonium bilaterally into the BLA (c), and significantly less time in the center, after microinjection of psalmotoxin into the BLA (d), compared with the time they spent in the center of the open field when injected with the vehicle. e, f, In the light/dark box test, rats microinjected with ammonium bilaterally into the BLA took a significantly longer time to enter the dark compartment (e), and spent more total time in the light compartment (f), compared with rats injected with the vehicle; **p < 0.01, ***p < 0.001.