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. 2018 Jan-Dec:14:1744806918804441.
doi: 10.1177/1744806918804441. Epub 2018 Sep 13.

Fear extinction learning ability predicts neuropathic pain behaviors and amygdala activity in male rats

Guangchen Ji  1   2 , Vadim Yakhnitsa  1 , Takaki Kiritoshi  1 , Peyton Presto  1 , Volker Neugebauer  1   2
Affiliations

Fear extinction learning ability predicts neuropathic pain behaviors and amygdala activity in male rats

Guangchen Ji et al. Mol Pain. 2018 Jan-Dec.

Abstract

Background The amygdala plays a key role in fear learning and extinction and has emerged as an important node of emotional-affective aspects of pain and pain modulation. Impaired fear extinction learning, which involves prefrontal cortical control of amygdala processing, has been linked to neuropsychiatric disorders. Here, we tested the hypothesis that fear extinction learning ability can predict the magnitude of neuropathic pain. Results We correlated fear extinction learning in naive adult male rats with sensory and affective behavioral outcome measures (mechanical thresholds, vocalizations, and anxiety- and depression-like behaviors) before and after the induction of the spinal nerve ligation model of neuropathic pain compared to sham controls. Auditory fear conditioning, extinction learning, and extinction retention tests were conducted after baseline testing. All rats showed increased freezing responses after fear conditioning. During extinction training, the majority (75%) of rats showed a decline in freezing level to 50% in 5 min (fear extinction+), whereas 25% of the rats maintained a high freezing level (>50%, fear extinction-). Fear extinction- rats showed decreased open-arm preference in the elevated plus maze, reflecting anxiety-like behavior, but there were no significant differences in sensory thresholds, vocalizations, or depression-like behavior (forced swim test) between fear extinction+ and fear extinction- types. In the neuropathic pain model (four weeks after spinal nerve ligation), fear extinction- rats showed a greater increase in vocalizations and anxiety-like behavior than fear extinction+ rats. Fear extinction- rats, but not fear extinction+ rats, also developed depression-like behavior. Extracellular single unit recordings of amygdala (central nucleus) neurons in behaviorally tested rats (anesthetized with isoflurane) found greater increases in background activity, bursting, and evoked activity in fear extinction- rats than fear extinction+ rats in the spinal nerve ligation model compared to sham controls. Conclusion The data may suggest that fear extinction learning ability predicts the magnitude of neuropathic pain-related affective rather than sensory behaviors, which correlates with differences in amygdala activity changes.

Keywords: Amygdala; anxiety; emotions; fear extinction; neuronal activity; pain; vulnerability.

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Figures

Figure 1.
Figure 1.
Interindividual differences in fear extinction learning ability in normal naive rats. Auditory fear conditioning (a), extinction (b), and extinction retention (d) tests were conducted using two distinct context chambers. (a) Day 1, rats were habituated to context A followed by fear conditioning (2 CS-US pairs, see Methods section). Diagram illustrates experimental protocol. Symbols in the line graph show freezing responses expressed in percent per 30-s block during fear conditioning with 2 CS-US pairings. (b) Day 2, rats were habituated to context B followed by extinction training (30 CSs, no US). Diagram illustrates experimental protocol. Symbols in the line graph show freezing responses to tone (CS) expressed in percent per 30-s block. (c) Bar histograms show distribution of rats with fear extinction (FE+) and rats with delayed and weak (freezing levels >50%) fear extinction (FE−). For details see Methods and Results section. (d) Day 3, rats were habituated to context B, followed by extinction retention testing (5 CSs, no US). Diagram illustrates experimental protocol. Symbols in the line graph show freezing response to tone (CS) expressed in percent per 30-s block. *, **, and *** indicate P 
Figure 2.
Figure 2.
Interindividual differences in pain-related behaviors of FE+ and FE− rats. (a and b) Duration (s) of audible and ultrasonic vocalizations, respectively, evoked by brief (15 s) noxious mechanical compression of the hind paw. Significant differences were found between FE+ (n = 11) and FE− (n = 13) neuropathic (SNL) rats but not between FE+ (n = 14) and FE− (n = 14) sham rats. *, **, and *** indicate P 
Figure 3.
Figure 3.
Comparison of behaviors before and after fear conditioning/extinction trials. Measurements were made in normal naive FE+ (n = 21) and FE− (n = 9) rats two days before (pre-FE) and two days after (post-FE) the fear conditioning/extinction trials. (a) Duration (s) of ultrasonic vocalizations evoked by brief (15 s) noxious mechanical compression of the hind paw. (b) Center duration in the OFT. (c) Mechanical thresholds measured with an electronic von Frey anesthesiometer. There were no significant differences between FE+ and FE− rats and between pre-FE and post-FE values in any of these tests (ANOVA; see Results section). Bar histograms show means ± SEM. OFT: open field test; FE: fear extinction. *, **, *** P
Figure 4.
Figure 4.
Examples of individual amygdala (CeLC) neurons in FE+ and FE− sham and SNL rats. (a and b) Top, peristimulus time histograms show the number of action potentials (spikes) per second for individual neurons. Innocuous (100 g/6 mm2) and noxious (500 g/6 mm2) stimuli (compression of the hind paw) are indicated by horizontal lines (15-s duration). Bottom, joint ISI plots (previous ISI against the subsequent ISI) show burst activity of individual neurons indicated by the rectangular insets (dots within the dashed red lines represent the first spike in a burst). Each dot represents a burst event in the individual neuron shown in each of the four plots. (a) Individual CeLC neurons recorded in sham FE+ rats and in sham FE− rats. (b) Individual CeLC neurons recorded in SNL FE+ rats and in SNL FE− rats. Individual CeLC neurons (8) from different experimental groups (4); one neuron recorded per animal. ISI, interspike interval; FE: fear extinction.
Figure 5.
Figure 5.
Sample of CeLC neurons in FE+ and FE− sham and SNL rats. Background activity (in the absence of any intentional stimulation) (a) and responses of CeLC neurons to brief (15 s) innocuous (b), and noxious (c) compression of the hind paw with a calibrated forceps. For net evoked responses, background activity (spikes/15 s) preceding the stimulus was subtracted from the total number of spikes during stimulation (15 s). (d) Mean number of bursts (see dots in Figure 4 meeting the burst definition) in a 5-min period of recording background activity of neurons in each experimental group. Bursts/s were calculated for each neuron by dividing the number of bursts by 300 s (n[burst]/300 s). Bar histograms show means ± SEM. Recordings were made in sham FE+ rats (n = 9 neurons), sham FE− rats (n = 7 neurons), SNL FE+ rats (n = 8 neurons), and SNL FE− rats (n = 9 neurons). *, **, and *** indicate P < 0.05–0.001, ANOVA with Bonferroni post hoc tests (see “Results” section). FE: fear extinction; SNL: spinal nerve ligation. *, **, *** P < 0.05, 0.01, 0.001.
Figure 6.
Figure 6.
Histologically verified recording sites of CeLC neurons in FE+ and FE− sham and SNL rats. Diagrams show coronal brain sections at different levels posterior to bregma (−2.30 to −2.80). The medial (CeM), lateral (CeL), and latero-capsular (CeLC) subdivisions of the central nucleus of the amygdala (CeA) are shown next to each section. Blue symbols, neurons recorded in FE+ rats and red symbols, neurons from FE− rats. Scale bars are 1 mm. FE: fear extinction; SNL: spinal nerve ligation.
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