Pavlovian fear memory induced by activation in the anterior cingulate cortex

Abstract

Identifying higher brain central region(s) that are responsible for the unpleasantness of pain is the focus of many recent studies. Here we show that direct stimulation of the anterior cingulate cortex (ACC) in mice produced fear-like freezing responses and induced long-term fear memory, including contextual and auditory fear memory. Auditory fear memory required the activation of N-methyl-D-aspartate (NMDA) receptors in the amygdala. To test the hypothesis that neuronal activity in the ACC contributes to unpleasantness, we injected a GABAA receptor agonist, muscimol bilaterally into the ACC. Both contextual and auditory memories induced by foot shock were blocked. Furthermore, activation of metabotropic glutamate receptors in the ACC enhanced behavioral escape responses in a noxious hot-plate as well as spinal nociceptive tail-flick reflex. Our results provide strong evidence that the excitatory activity in the ACC contribute to pain-related fear memory as well as descending facilitatory modulation of spinal nociception.

Figure 1

ACC stimulation induces ultrasonic vocalization in freely moving mice. (a) An example of ultrasonic responses from a single mouse at four different frequencies before, during and after ACC stimulation (at 0.3 mA). 1 min duration; see filled circle for the stimulation site within the ACC in (e); (b) ACC stimulation (0.3 mA; n = 6) increased the frequency of individual ultrasonic responses; * P <0.05, comparing the frequency during ACC stimulation with baseline response before the stimulation; (c) ACC stimulation (0.3 mA; n = 6) also increased the duration of single ultrasonic response; * P < 0.05, comparing the duration during ACC stimulation with baseline duration before the stimulation; (d) Summarized data of ACC stimulation (n = 6) produced ultrasonic responses at different intensities. Total vocalization responses (sec) within 2 min ACC stimulation were plotted against the intensity of stimulation; (e) Stimulating sites in ACC on the schematic representation of coronal section 0.62 mm anterior to the Bregma. Filled circle, for data shown in (a); open circles, other sites for data shown in (b-d).

Figure 2

ACC stimulation induces long-term fear memory. (a) Three pairings of 30 s tone and 10 s electrical train stimulation were delivered in the paired group on the conditioning day; (b,c) Percentage freezing to the tone (b) and the conditioning context (c) measured at 1 hr, 1 day and 3 days after paired training. After paired training, long-lasting fear memory was detected in most of the mice (n = 16 mice, filled circles), while some other mice showed no freezing across the test periods (n = 5, data not shown). After unpaired training of tone and ACC stimulation, mice showed no freezing to the tone (n = 6, open squares) but clear memory to the training environment; * P < 0.05 compared with the unpaired group;(d) Stimulating sites in ACC on the schematic representation of coronal section 0.62 mm anterior to the Bregma. Filled circles, effect sites of ACC paired group; open circles, no effect sites of ACC paired group; open squares, ACC unpaired group.

Figure 3

Activation of metabotropic glutamate receptors in the ACC caused long-term fear memory. (a) ACC tACPD microinjection (0.25 μg in 0.5 μl; indicated by an arrow) was paired with a tone (indicated by filled bar) on the conditioning day; (b, c) Percentage freezing to the tone (b) and the conditioning context (c) measured at 1 day and 3 days after paired training (n = 5 mice). *P < 0.05 compared with the control. (d) The sites in the ACC for tACPD microinjection on the schematic representation of coronal section 0.62 mm anterior to the Bregma.

Figure 4

Primary somatosensory cortex (S1) stimulation produces no long-term fear memory. (a, b) Three pairings of 30 s tone and 10 s electrical train stimulation were delivered to somatosensory cortex (S1) caused neither long-term auditory (a) nor contextual memory (b) (n = 5). (c) Sites for stimulation in the S1.

Figure 5

NMDA receptors in the amygdala is required for auditory fear memory induced by ACC stimulation. (a) Bilateral microinfusions of AP-5 (2 μg/μl, 0.5 μl/side) in the BLAC 15 min before conditioning impaired auditory fear memory but no effect on contexual fear memory when tested 1 day later. Filled bars, AP-5 group (n = 5); open bars, saline group (n = 4). * P < 0.05 compared with saline-injected group. (b) Stimulation sites in ACC. Filled circles, AP-5 group; open circles, saline group. (c) Microinjection sites in the BLAC on the schematic representation of coronal section 1.94 mm posterior to the Bregma. Filled circles, AP-5 group; open circles, saline group.

Figure 6

ACC inactivation by a GABA_A receptor agonist impairs fear memory by foot shock. (a,b) Mice receiving muscimol microinjection (1 μg/μl, 0.5 μl/each side, n = 6 mice, black bars) into bilateral ACC 15 min before conditioning showed reduced auditory (a) and contextual (b) fear memory induced by classic foot shock conditioning). * P < 0.05 compared with the saline treated group (n = 6, open bars). (c) Microinjection sites in the ACC. Filled circles: muscimol group; open circles, saline group.

Figure 7

Activation of mGluRs in the ACC facilitated escaping behavioral responses in a hot-plate. (a) A diagram explaining a new behavioural escape test using the modified hot-plate instrument. (b) Mice receiving tACPD microinjection into the unilateral ACC 10 min before training showed faster escape response. * P < 0.05 compared with the control group (n = 6 mice, open squares). (c) Extinction responses in control and tACPD-treated groups were similar. MPE were calculated as: (response latency – baseline latency)/(180 – baseline latency). 100% MPE indicates that mouse stayed in the same plate for 3 min without moving into the safe area.

Figure 8

Spinal serotonin receptors contribute to descending facilitatory modulation from the ACC. (a, b) Microinjection of tACPD (0.25 μg in 0.5 μl) in the ACC facilitated the hot-plate responses (a, i.e., reduced response latency) and spinal nociceptive tail-flick reflex (b) in freely moving mice; (c, d) In freely moving (c) or anesthetized rats (d), tACPD microinjection in the ACC also facilitated the tail-flick reflex. Intrathecal injection of a serotonergic receptor antagonist methysergide (32.0 nmoles/10 μl) at a dose that blocked descending facilitation also blocked the facilitation of the tail-flick reflex.(e) A model explaining the neuronal pathways, which contribute to ACC activation, produced fear memory and descending facilitatory modulation of spinal nociception.

Figure 9

Fear conditioning did not cause long-term plasticity in ACC-ACC synapses. (a) Evoked fast responses in the ACC by stimulation applied to the other side of ACC were not affected by single foot shock that induced classic fear memory. However, TBS induced synaptic potentiation in the ACC. Insets: Traces of evoked responses before, 3 hour, 1 day and 3 days after fear conditioning.(b) Summarized data of experiments shown in (a) and mice receiving the treatment without the foot shock (n = 2 mice).