Neuronal correlates of fear in the lateral amygdala: multiple extracellular recordings in conscious cats

Abstract

Much data implicates the amygdala in the expression and learning of fear. Yet, few studies have examined the neuronal correlates of fear in the amygdala. This study aimed to determine whether fear is correlated to particular activity patterns in the lateral amygdaloid (LA) nucleus. Cats, chronically implanted with multiple microelectrodes in the LA and a catheter in the femoral artery, learned that a series of tones interrupted by a period of silence (5 sec) preceded the administration of a footshock. During the silent period, their blood pressure increased, indicating that they anticipated the noxious stimulus. In parallel, the firing rate of LA neurons doubled, and the discharges of simultaneously recorded cells became more synchronized. Moreover, cross-correlation of focal LA waves revealed a significant increase in synchrony restricted to the theta band. In keeping with this, perievent histograms of neuronal discharges revealed rhythmic changes in the firing probability of LA neurons in relation to focal theta waves. Finally, the responsiveness of LA cells to the stimuli predicting the footshock (the tones) increased during the trials, whereas responses to unrelated stimuli (perirhinal shocks) remained stable. Thus, during the anticipation of noxious stimuli, a state here defined anthropomorphically as fear, the firing rate of LA neurons increases, and their discharges become more synchronized through a modulation at the theta frequency. The presence of theta oscillations in the LA might facilitate cooperative interactions between the amygdala and cortical areas involved in memory.

Fig. 1.

Anticipatory increases in blood pressure and heart rate in conditioned animals. A, Experimental paradigm. A series of 1 sec tones, interrupted by a period of silence (5 sec), was presented to the animals (4 tones, silence, 2 tones). In control conditions, no noxious stimuli were administered. In later sessions, an electrical shock to the front paws (0.5 sec, 1.5 mA) was administered, 0.5 sec after the onset of the last tone (A, arrow). The vertical lines in B–D indicate the tone onsets. The changes in arterial blood pressure (B) and heart rate (C) observed during the tones are depicted for naive animals (B, C, lines without symbols) and for conditioned animals (B, C, lines with symbols; average of three experiments, in three cats). In B, the systolic (top curves) and diastolic (bottom curves) blood pressure are shown. The average interbeat interval, as estimated in C, was used to scale the blood pressure and heart rate data in time. For clarity, only the SE of the data obtained after conditioning is shown in B and C. D depicts two superimposed trials obtained in the same cat before and after conditioning.

Fig. 2.

Histological determination of recording and stimulating sites. Thionin-stained coronal sections arranged from rostral in A to caudal in D.Arrowheads in A–C point the electrolytic lesions (0.5 mA for 5 sec) made at the end of the experiments to mark selected recording sites in the LA.D, Stimulating electrodes in the perirhinal cortex. Scale bar in millimeters. BL, Basolateral nucleus of the amygdala; CE, central nucleus; CL, claustrum; GP, globus pallidus; H, hippocampal formation; IC, internal capsule;LA, lateral nucleus of the amygdala; OT, optic tract; PU, putamen; rh, rhinal sulcus; V, ventricle.

Fig. 3.

Physiological identification of LA projection cells by antidromic invasion from the perirhinal cortex. (A, B, C1) Three simultaneously recorded LA neurons that generated antidromic spikes in response to electrical stimuli applied in the perirhinal cortex.Arrowheads point to stimulation artifacts. In each case, several responses are superimposed. Note fixed response latencies.C2, Response of the cell shown in C1 to three perirhinal stimuli delivered at 300 Hz. Note the ability of the antidromic spikes to follow high-frequency stimulation. Small arrows in B and C2 point to traces where the antidromic spike failed. The neuron inA was located in the caudolateral part of the LA, whereas those in B and C were located 2 mm more rostrally, and at different lateromedial levels (C 0.8 mm more lateral than B). Data were digitally filtered (100 Hz to 10 kHz).

Fig. 4.

Increases in the firing rate and auditory responsiveness of LA neurons during the anticipation of noxious stimuli. Instantaneous firing rate of LA neurons in naive (A, n = 36) and conditioned (B, n = 49) animals (bins of 100 msec) during the presentation of the tones (T1–T6, thin vertical lines). The tone series was presented four times. For each cell, the spike counts in the four trials were averaged. Then, the activity of all the cells within a group (control or conditioned) was averaged and converted into instantaneous firing rates. Theright part of the figure shows the average field potentials evoked by the six tones in naive (C) and conditioned (D) animals. Each wave represents the average of 144 responses (36 sites times 4 trials) inC and 196 responses (49 sites times 4 trials) inB. Negative, downward. The amplitude of tone-evoked field potentials was measured by subtracting the peak voltage (within a SE of the average latency) from the average voltage value of the 50 msec preceding the tone onset (dashed lines).

Fig. 5.

LA responsiveness to perirhinal stimuli does not increase during the anticipation of noxious stimuli. Perirhinal-evoked LA responses in the same cat before (A) and after (B) introduction of the noxious stimulus (trials 1 and 6 of the first conditioning session, respectively). Same recording site, neuron, and stimulation intensity in Aand B. 1, Window discriminator output.Thick vertical line indicates perirhinal stimuli.2, 3, Peristimulus histogram of neuronal discharges and simultaneously recorded evoked potential (averages of 12 and 6 in2 and 3, respectively). R, Responsiveness (number of spikes divided by number of shocks). Average unit responsiveness and field potential amplitude were 1.50 ± 0.151 and 218.2 ± 16.86 μV in A2, 1.83 ± 0.167 and 245 ± 22.53 μV in A3, 2.25 ± 0.131 and 327.3 ± 18.11 μV in B2, and 2.5 ± 0.342 and 336.4 ± 27.43 μV in B3.C, Normalized amplitude of field potentials evoked by perirhinal stimuli in three conditioned cats in quiescent periods (black bars) and during the anticipation of noxious stimuli (white bars). Whereas the amplitude of the field potentials and neuronal responsiveness did not change significantly within a trial (A2 vs A3 orB2 vs B3), it increased from the naive to the conditioned state (A vs B).

Fig. 6.

Increased synchrony in the activity of LA neurons during the anticipation of noxious stimuli. In conditioned animals, cross-correlograms of neuronal discharges were computed for 59 pairs of simultaneously recorded LA neurons before the tones (A) or during the silent period preceding the noxious stimulus (B). Before averaging, the individual cross-correlograms were normalized to the number of spikes generated by the reference cell. The number of cell couples (n) as well as the number of spikes generated by the reference (nR) and test (nT) cells are indicated on the top right of the histograms.

Fig. 7.

Simultaneously recorded LA sites exhibit increased correlation in the theta band during the anticipation of noxious stimuli. A, Cross-correlograms of local field potentials recorded simultaneously in two LA sites (distance, 0.8 mm) before the tones (A1) and during the silent period (A2). Focal waves were digitally filtered from 2 to 55 Hz and divided in 1 sec segments sliding in steps of 100 msec over the 5 sec epoch preceding the tones or the silent period. Then, the corresponding wave segments were cross-correlated. Only 10 of the resulting cross-correlograms are shown in A1 andA2. A3, Power spectrum of cross-correlograms before the tones (thin line) and during the silent period (thick line). To compute this, the analysis described in A1 and A2 was repeated for all pairs of simultaneously recorded LA sites (n = 59), and the FFTs of the resulting cross-correlograms were averaged. B, Population perievent histograms of LA firing using the positive peaks of focal theta waves as reference times, before the tones (B1) or during the silent period (B2). Average of 49 perievent histograms. To compute these histograms, focal waves were digitally filtered (4–7 Hz), and the positive peaks of theta waves exceeding 1.5 times the SD of the 5 sec segments were detected. The number of theta peaks and spikes were 803 and 652 for B1 compared to 1076 and 1986 for B2.