Fast oscillatory activity induced by kainate receptor activation in the rat basolateral amygdala in vitro

Abstract

The basolateral amygdala (BLA) has a fundamental role in affective processing. In vivo studies have revealed rhythmic population activity of a similar type to that seen in the hippocampus and cortical areas during learning tasks. The amygdala contains densely interconnected networks of inhibitory interneurons similar to those responsible for fast network activity generation in the hippocampus and other cortical structures. Here we report that neuronal networks of the BLA in isolation generate persistent, gamma frequency (30-80 Hz) oscillations upon kainate receptor activation with kainic acid. We show that, like other cortical structures, BLA oscillations are completely dependent upon γ-aminobutyric acid (GABA)ergic inhibition. GABA(A) receptor blockade abolished all oscillations, and the activity was also sensitive to the barbiturate, pentobarbital. Blockade of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors or N-methyl-D-aspartate (NMDA) receptors had no significant effect on gamma activity. However, the GluR5-containing kainate receptor-specific antagonist (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxybenzyl) pyrimidine-2,4-dione (UBP302) abolished oscillations-evidence that glutamatergic receptor involvement is predominantly kainate receptor mediated. The mixed AMPA/kainate receptor antagonist 6-nitro-7-sulphamoylbenzo[f]quinoxalone-2,3-dione disodium (NBQX) abolished all oscillatory activity in 8/14 of slices tested. In the remaining slices, gamma frequency activity was abolished to reveal a low-amplitude, NMDA receptor-dependent, beta frequency (10-20 Hz) oscillation. Gamma oscillations are abolished by gap junction blockade. While these data show the BLA capable of generating gamma rhythms in common with other cortical areas studied to date, the network mechanisms appear to be different, suggesting a unique network structure underlies amygdala rhythmogenesis. Understanding how BLA networks produce synchronous activity is paramount to understanding how the BLA executes influence on important cognitive processes such as emotional learning.

FIG. 1

In the presence of KA (200–400 nm), oscillatory activity of gamma frequency builds up in the BLA. (A) Sample trace of local field potential activity recorded extracellularly in the BLA in control aCSF and after the application of kainate to the aCSF. (B) Spectrogram produced using 60-s epochs of gamma activity to illustrate stable frequency of BLA gamma oscillation. (C) Pooled power spectra corresponding to traces in control (grey) and in the presence of kainate (black; n = 6). Scale bars: 250 μV, 100 ms (A).

FIG. 2

GABA_A receptor-mediated IPSPs are critical for oscillatory activity in BLA networks. (A) Example extracellular (ec) traces of field gamma oscillation in the BLA in control conditions and in the presence of gabazine (1 μm). The corresponding power spectra, control (black) and gabazine (grey), demonstrates the sensitivity of the BLA gamma oscillation to antagonism of the GABA_A receptor. KA, kainic acid. (B) At resting membrane potential, the BLA principal neuron was identified via responses to hyperpolarising and depolarising voltage steps. The cell was filled in recording to illustrate its location in the BLA. Scale bar: 100 μm. (C) Intracellular recording (ic) of cell with membrane potential held at −20 mV to reveal the IPSPs the cell is receiving during kainite-induced gamma oscillation and in the presence of gabazine. Cross-correlogram demonstrated the degree of correlation between extracellular and intracellular (principal cell IPSPs) oscillatory activity in the control gamma oscillation (black) and in the presence of gabazine (grey). Scale bars: 50 μV, 100 ms (A); 20 mV, 100 ms (B); 2 mV, 100 ms (C).

FIG. 3

Pentobarbital reduces the frequency and amplitude of BLA gamma oscillations by modulation of BLA principal neuron IPSPs. (A) Example traces of extracellular recording (ec) of field gamma oscillation in the BLA in control gamma oscillation and in the presence of the barbituate, pentobarbital. The corresponding power spectra, control (black) and pentobarbital (grey), demonstrate the sensitivity of the BLA gamma oscillation to prolongation of the GABA_A receptor kinetics. KA, kainic acid. (B) Intracellular recording of cell with membrane potential held at −20 mV to reveal the IPSPs the cell is receiving in control and pentobarbital. Example traces illustrate the reduction in amplitude and increase in decay time of gamma frequency BLA principal neuron IPSPs produced by pentobarbital. Cross-correlogram [field (black) vs. principal cell IPSPs (grey)] to show the correlation between oscillatory activity and the inhibitory potentials the cell is receiving in control and pentobarbital. Scale bars: 100 μV, 100 ms (A); 5 mV, 100 ms (B); 10 mV, 20 ms (B).

FIG. 4

AMPA receptors have no critical role in gamma oscillations in the BLA. Application of the AMPA receptor-specific antagonist SYM 2206 (50 μm) had no significant effect on gamma oscillations in the BLA. (A) Example extracellular traces of gamma frequency oscillations induced by kainate and in the presence of the AMPA receptor antagonist, SYM 2206. (B) Corresponding pooled power spectra showing a lack of alteration in BLA gamma oscillation (black) power and frequency following AMPA receptor blockade (grey). Scale bars: 200 μV, 100 ms (A).

FIG. 5

BLA gamma oscillations are sensitive to AMPA/kainate receptor antagonism. (A) Example extracellular field (ec) traces illustrating control BLA gamma oscillations and the abolition of this activity in the presence of NBQX (20 μm) and corresponding pooled power spectra. KA, kainic acid. (B) Example extracellular field (ec) traces illustrating control BLA gamma oscillations and the abolition of this activity in the presence of UBP302 (25 μm) and corresponding pooled power spectra. (C) Sample traces of concurrent intracellular IPSPs (ic) during BLA gamma oscillation and cross-correlation analysis of ec and ic recordings that demonstrates that correlation between ec and ic activity is abolished with NBQX. Scale bars: 200 μV, 100 ms (A and B); 2 mV and 100 ms (C).

FIG. 6

AMPA/kainate receptor antagonism can in some cases reveal a residual NMDA receptor-dependent beta oscillation. (A) Example extracellular field traces (ec) illustrate the emergence of BLA beta oscillatory activity in the presence of NBQX, and subsequent abolition of beta oscillation with d-APV. Corresponding pooled power spectra for these experiments. KA, kainic acid. (B) Sample traces of concurrent intracellular IPSPs (ic) in all experimental conditions. Associated cross-correlation analysis of ec and ic recordings demonstrates the reduction (NBQX, beta oscillation) and abolition (d-APV) between BLA ec and ic oscillations. Scale bars: 200 μV, 100 ms (A); 2 mV, 100 ms (B).

FIG. 7

NMDA receptor blockade alone has no effect on gamma oscillations in the BLA. Sample extracellular traces show the lack of sensitivity of BLA gamma oscillations to NMDA receptor antagonism and subsequent reduction by NBQX. These data are summarised in the corresponding pooled power spectra for the three conditions control (black), d-APV (grey) and NBQX (light grey). Scale bars: 200 μV, 100 ms (A). KA, kainic acid.

FIG. 8

Gap junctions are required for persistent gamma oscillations in the BLA. (A) Example traces of extracellular field recording (ec) of gamma oscillation in the BLA in control and in the presence of the broad gap junction blocker, octanol (1 mm). Corresponding pooled power spectra illustrate the abolition of BLA gamma oscillation by gap junction antagonism. Example traces from BLA principal neurons in response to depolarising (+0.4 nA) steps in the absence and presence of octanol demonstrate a lack of effect on cellular excitability by the gap junction blocker. KA, kainic acid. (B) Sample traces of concurrent intracellular IPSPs (ic) in the presence and absence of the gap junction blocker. Associated cross-correlation analysis of ec and ic recordings demonstrates the disruption of synchrony between BLA ec and ic oscillations in the presence of octanol. Scale bars: 100 μV, 100 ms; 10 mV, 200 ms (A); 10 mV, 100 ms (B).