*Article* **Fear Extinction-Based Inter-Individual and Sex Differences in Pain-Related Vocalizations and Anxiety-like Behaviors but Not Nocifensive Reflexes**

**Peyton Presto <sup>1</sup> , Guangchen Ji 1,2, Riley Junell <sup>1</sup> , Zach Griffin <sup>1</sup> and Volker Neugebauer 1,2,3,\***

	- Center, Lubbock, TX 79430-6592, USA

**Abstract:** Inter-individual and sex differences in pain responses are recognized but their mechanisms are not well understood. This study was intended to provide the behavioral framework for analyses of pain mechanisms using fear extinction learning as a predictor of phenotypic and sex differences in sensory (mechanical withdrawal thresholds) and emotional-affective aspects (open field tests for anxiety-like behaviors and audible and ultrasonic components of vocalizations) of acute and chronic pain. In acute arthritis and chronic neuropathic pain models, greater increases in vocalizations were found in females than males and in females with poor fear extinction abilities than females with strong fear extinction, particularly in the neuropathic pain model. Female rats showed higher anxiety-like behavior than males under baseline conditions but no inter-individual or sex differences were seen in the pain models. No inter-individual and sex differences in mechanosensitivity were observed. The data suggest that vocalizations are uniquely suited to detect inter-individual and sex differences in pain models, particularly in chronic neuropathic pain, whereas no such differences were found for mechanosensitivity, and baseline differences in anxiety-like behaviors disappeared in the pain models.

**Keywords:** vocalizations; fear extinction; pain; sex differences

### **1. Introduction**

Inter-individual and sex differences have been well documented with regard to anxiety- and depression-like conditions [1–3] and in pain [4–6]. However, neural mechanisms and biomarkers related to pain vulnerability and resilience, including potential sexual dimorphisms, have yet to be fully elucidated. Intricate interactions of sensory, cognitive, and emotional-affective dimensions form the highly complex and intense experience of pain. The strong negative affective component of pain presents a challenge for effective therapeutic strategies, as patients suffering from chronic pain are at increased risk of developing mood and anxiety disorders, and vice versa [7–10]. This suggests that pain may share neurobiological mechanisms, including emotional network neuroplasticity, with negative emotions such as fear [11,12]. Fear learning and extinction networks have been implicated in neuropsychiatric disorders such as anxiety disorders, post-traumatic stress disorder (PTSD), and obsessive compulsive disorder (OCD) [13–15]. Vulnerability to these disorders has been predicted using fear extinction (FE) learning ability as a biomarker for inter-individual differences in the preclinical [16] and clinical [17] setting.

Behavioral studies are a crucial tool for the validation of pain mechanisms and for the assessment of potential pharmacological therapies. A variety of behavioral methods have been developed in preclinical pain models for the evaluation of traits pertaining to

**Citation:** Presto, P.; Ji, G.; Junell, R.; Griffin, Z.; Neugebauer, V. Fear Extinction-Based Inter-Individual and Sex Differences in Pain-Related Vocalizations and Anxiety-like Behaviors but Not Nocifensive Reflexes. *Brain Sci.* **2021**, *11*, 1339. https://doi.org/10.3390/ brainsci11101339

Academic Editor: Stefan M. Brudzynski

Received: 14 September 2021 Accepted: 6 October 2021 Published: 11 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

sensorimotor function, anxiety- and depressive-like behavior, social interactions, cognitive function, and emotional-affective responses [18]. Higher integrated pain behavior at supraspinal levels has been assessed using vocalizations. Vocalizations are an important method of communication among rodents [19], with frequencies in the audible and ultrasonic ranges. Audible vocalizations of rats in response to a noxious stimulus indicate a nociceptive reaction, whereas ultrasonic vocalizations of the 22 kHz type represent negative emotional-affective responses [20,21]. Ultrasonic vocalizations are considered an effective indicator for measuring negative emotional status and have been used in different experimental models of pain, including arthritis pain [21–24], chronic cancer pain [25,26], and neuropathic pain [27–30]. However, some have called into question the reliability of vocalizations in assessing pain-related behavior [31] and others have found that vocalizations may occur as a response to handling [32]. While a valuable behavioral measure, vocalizations as a pain assessment may be most informative when used in combination with other pain indicators [33]. Inter-individual and sex differences in audible and ultrasonic vocalizations, particularly in the context of pain and fear interactions, have not been determined.

The purpose of this study was to examine the predictive value of fear extinction (FE) learning ability for inter-individual differences in pain-related behavioral responses, particularly emotional-affective pain aspects, with regard to sex. We subjected adult male and female rats to cued fear learning and FE tests and correlated inter-individual differences with pain responses in models of acute arthritis pain and chronic neuropathic pain. We also investigated sex differences in FE phenotypes for measures of sensory (mechanical withdrawal thresholds) and emotional-affective (open field tests for anxiety-like behaviors and audible and ultrasonic components of vocalizations) pain-related behaviors.

#### **2. Materials and Methods**

#### *2.1. Animals*

A total of 215 male and 190 female Sprague-Dawley rats (150–350 g, 6–12 weeks of age) were group-housed in a temperature-controlled room under a 12 h light/dark cycle with unrestricted access to food and water. On each experimental day, rats were transferred from the animal facility and allowed to acclimate to the laboratory for at least 1 h. Experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC; protocol #14006) at Texas Tech University Health Sciences Center and conformed to the guidelines of the International Association for the Study of Pain (IASP) and of the National Institutes of Health (NIH).

### *2.2. Experimental Protocol*

Naïve rats were subjected to fear conditioning and FE trials. Rats were then randomly assigned to the arthritis pain model (see Section 2.3) or the neuropathic pain model (see Section 2.4). One week later, the neuropathic pain model was induced or sham surgery was performed. Four weeks after surgery, neuropathic pain-related behavioral changes reach a stable plateau in this model [29]. The arthritis pain model was induced in a separate group of rats at the four-week time point to achieve age-matched experimental groups. Behavioral studies were performed four weeks after surgery or 6 h after arthritis induction when behavioral and neurobiological changes are known to reach a maximum plateau [21]. The experimenter was blinded with regard to the FE phenotype, the neuropathic versus sham condition, and the arthritis versus untreated control condition. The experimental design is illustrated in Figure 1.

**Figure 1.** Experimental design. Rats underwent fear conditioning and extinction learning protocols before being separated into FE+ and FE− groups for either the acute arthritis pain (vs. untreated control) groups or the chronic neuropathic pain (vs. sham control) groups. FE: fear extinction. **Figure 1.** Experimental design. Rats underwent fear conditioning and extinction learning protocols before being separated into FE+ and FE− groups for either the acute arthritis pain (vs. untreated control) groups or the chronic neuropathic pain (vs. sham control) groups. FE: fear extinction.

#### *2.3. Arthritis Pain Model 2.3. Arthritis Pain Model*

The well-established mono-arthritis pain model mimics the acute phase of the human osteoarthritis condition and was induced in the left knee joint as described in detail previously [21]. Rats were briefly anesthetized with isoflurane (2–3%; precision vaporizer, Harvard Apparatus, Holliston, MA) and a kaolin suspension (4% in sterile saline, 100 μL) was slowly injected into the joint cavity followed by repetitive flexions and extensions of the leg for 15 min. A carrageenan solution (2% in sterile saline, 100 μL) was then injected into the knee joint cavity and the leg was flexed and extended for another 5 min. This treatment paradigm (the K/C arthritis model) reliably produces a localized inflammation in only one knee joint with damage to the cartilage within 1–3 h. K/C arthritis persists for at least a week and is associated with pain behaviors and neural activity changes in the central and peripheral nervous system. Naïve rats that underwent similar handling but did not receive intraarticular injections were used as a control group, as data from our previous studies demonstrated no differences in the behavior of untreated rats and of those that received intraarticular saline injection [34] or needle insertion [35]. This justified the use of naïve rats as an appropriate control for the K/C pain model, which is well established in our laboratories [36–38]. The well-established mono-arthritis pain model mimics the acute phase of the human osteoarthritis condition and was induced in the left knee joint as described in detail previously [21]. Rats were briefly anesthetized with isoflurane (2–3%; precision vaporizer, Harvard Apparatus, Holliston, MA) and a kaolin suspension (4% in sterile saline, 100 µL) was slowly injected into the joint cavity followed by repetitive flexions and extensions of the leg for 15 min. A carrageenan solution (2% in sterile saline, 100 µL) was then injected into the knee joint cavity and the leg was flexed and extended for another 5 min. This treatment paradigm (the K/C arthritis model) reliably produces a localized inflammation in only one knee joint with damage to the cartilage within 1–3 h. K/C arthritis persists for at least a week and is associated with pain behaviors and neural activity changes in the central and peripheral nervous system. Naïve rats that underwent similar handling but did not receive intraarticular injections were used as a control group, as data from our previous studies demonstrated no differences in the behavior of untreated rats and of those that received intraarticular saline injection [34] or needle insertion [35]. This justified the use of naïve rats as an appropriate control for the K/C pain model, which is well established in our laboratories [36–38].

#### *2.4. Neuropathic Pain Model*

*2.4. Neuropathic Pain Model*  The well-established spinal nerve ligation (SNL) model of neuropathic pain [39] was used, which creates stable and long-lasting neuropathic pain behaviors. Rats were anesthetized with isoflurane (2–3%; precision vaporizer, Harvard Apparatus) and underwent sterile surgery where the left L5 spinal nerve was exposed and tightly ligated using 6–0 sterile silk. In the sham-operated control group, the nerve was exposed but not ligated. The well-established spinal nerve ligation (SNL) model of neuropathic pain [39] was used, which creates stable and long-lasting neuropathic pain behaviors. Rats were anesthetized with isoflurane (2–3%; precision vaporizer, Harvard Apparatus) and underwent sterile surgery where the left L5 spinal nerve was exposed and tightly ligated using 6–0 sterile silk. In the sham-operated control group, the nerve was exposed but not ligated.

#### *2.5. Behaviors*

#### *2.5. Behaviors*  2.5.1. Fear Conditioning and Extinction

2.5.1. Fear Conditioning and Extinction Fear conditioning and extinction learning tests were conducted using two chambers of a near infrared Video Fear Conditioning System (Med Associates Inc., Fairfax, VT, USA) as described previously [40–42]. The conditioning chambers were located inside a sound-Fear conditioning and extinction learning tests were conducted using two chambers of a near infrared Video Fear Conditioning System (Med Associates Inc., Fairfax, VT, USA) as described previously [40–42]. The conditioning chambers were located inside a sound-attenuating isolation cabinet with a metal grid flooring that was connected to a

grid stimulator to administer aversive foot shocks. Two distinct chambers with separate visual, olfactory, tactile, dimensional, and lighting environments were used (context A: white light, no fan in chamber, metal grid on chamber floor, lights on in experimental room, rat transported to chamber in transparent box, chamber cleaned with 50% ethanol; context B: near-infrared (NIR) light, fan on in chamber, flat chamber floor, lights off in experimental room, rat transported to chamber in opaque box, chamber cleaned with 70% isopropanol, colored insert with 3 drops of peppermint oil added to alter olfactory environment and physical dimensions). Day 1 consisted of the training phase where rats were habituated to the training chamber (context A) and allowed to explore freely for 5 min, followed by fear conditioning that consisted of a foot shock (0.7 mA, 2 s; the unconditioned stimulus, USA) delivered during the final 2 s of an auditory stimulus (white noise, 80 dB, 4.5 kHz, 30 s; the conditioned stimulus, CS). Two CS-US pairings were used (intertone interval, ITI, 120 s). On day 2, rats were placed in a different chamber (context B) and were habituated for 5 min, followed by extinction training (30 CSs, ITI 5 s). A mounted video camera in the conditioning chambers was used to record the behavior of each rat. Freezing behavior (expressed as a percentage of each 30 s period) was analyzed and quantified using Video Freeze software (Med Associates Inc.) as the conditioned response. Based on their FE learning ability, rats were classified into strong (FE+), "normal" (FE+/−), and weak (FE−) FE groups as determined by evidence of diminishing (below 50%) freezing responses during Phase I (before 600 s), Phase II (600–900 s), or Phase III (after 900 s) of extinction training (see the "Results" section for details). Rats in the two extreme groups (FE+ and FE−) were selected for further behavioral testing and randomly assigned to groups in the arthritis pain model (untreated FE+, untreated FE−, arthritis FE+, and arthritis FE−) or in the neuropathic pain model (sham FE+, sham FE−, SNL FE+, and SNL FE−). Four weeks after SNL or sham surgery, or 6 h after arthritis induction in an age-matched model, behavioral assays (see next paragraphs) were performed.

#### 2.5.2. Mechanosensitivity

Rats were briefly anesthetized with isoflurane (2–3%; precision vaporizer, Harvard Apparatus) and were placed slightly restrained in a customized recording chamber that permitted access to the hindlimbs (U.S. Patent 7,213,538) for stable testing. Hindlimb withdrawal thresholds were evaluated after recovery from anesthesia and after habituation to the recording chamber for 30 min. Hindlimb withdrawal thresholds were evaluated using calibrated forceps with a force transducer whose output was displayed in grams on an LED screen. The calibrated forceps were used to gradually compress the left knee joint (arthritis pain model) or the left hindpaw (neuropathic pain model) with a continuously increasing intensity until a withdrawal reflex was evoked as described in our previous studies [21,35,37,38,43–45]. The withdrawal threshold, defined as the force required to evoke a reflex response, was calculated using the average value from 2 to 3 trials.

#### 2.5.3. Emotional Responses

Components of vocalizations in the audible (20 Hz–16 kHz) and ultrasonic (25 ± 4 kHz) ranges were simultaneously measured after hindlimb withdrawal assays using an automatic computerized vocalization system consisting of a full-spectrum USB ultrasound microphone (max sampling rate: 384 kHz) and UltraVox XT four-channel recording and analysis system (Noldus Information Technology, Leesburg, VA, USA). Rats were briefly anesthetized with isoflurane (2–3%; precision vaporizer, Harvard Apparatus) and placed in the customized recording chamber for stable recordings of vocalizations evoked by natural stimulation. After the rat recovered from anesthesia and habituated to the recording chamber for 30 min, hindlimb withdrawal thresholds were evaluated (see Section 2.5.2) and the calibrated forceps with a force transducer were used for vocalization assays. Vocalizations were evoked by a brief (10 s), continuous noxious stimulus applied to the left knee joint (arthritis pain model; stimulus: 1500 g/30 mm<sup>2</sup> ) or to the left hindpaw (neuropathic pain model; stimulus: 500 g/6 mm<sup>2</sup> ) as described in our previous studies [20,28,29,37,38,42]. Vocalizations were automatically detected for 1 min and total durations of audible and ultrasonic components of vocalizations following the onset of mechanical stimulus were analyzed using UltraVox 3.2 software (Noldus Information Technology). For vocalization analyses, audible calls were labeled using frequency ranges of 20 Hz–16 kHz and ultrasonic components of calls were labeled using frequency ranges of 21–29 kHz. The following call descriptions were also specified: minimum amplitude, 50 units; minimum duration, 1 ms; maximum duration, 2000 ms; minimum gap between calls, 1 ms. Calls that fit these criteria were detected for each recording. At the conclusion of each experiment, call statistics for each recording were exported as a text file. The duration (in ms) for each individual call was summed for each 1 min recording period to give the total duration of audible and ultrasonic components of vocalizations for each rat.

#### 2.5.4. Anxiety-Like Behavior

Animal movements within the open field test (OFT) were used to measure anxiety-like behavior. Exploratory behavior in the central or peripheral zones of an arena (70 cm × 70 cm) with acrylic walls (height, 45 cm) was recorded for 15 min using a computerized video tracking and analysis system (EthoVision XT 11 software, Noldus Information Technology) as described previously [42,46]. Time spent in the center of the arena (35 cm × 35 cm) was calculated during the first 5 min. Avoidance of the center of the arena is interpreted to suggest anxiety-like behavior [42,46–48].

#### *2.6. Statistical Analysis*

All averaged values are presented as the mean ± SE. Statistical significance was accepted at the level *p* < 0.05. GraphPad Prism 9.0 software was used for all statistical analyses. Statistical analyses were performed on the raw data. For multiple comparisons, a two-way analysis of variance (ANOVA) was used with Bonferroni post hoc tests.

#### **3. Results**

#### *3.1. Inter-Individual and Sex Differences in FE Learning Ability of Naïve Male and Female Rats*

Fear learning and FE are well-established models of aversive learning that have been used to correlate behavior with neural structure and function, which involve cortico-limbic circuits centered on the amygdala [15]. We previously reported that the identification of distinct behavioral phenotypes based on FE ability in naïve male rats can serve as a predictor for inter-individual differences in pain sensitivity and amygdala neuronal activity in chronic neuropathic pain [42]. Here, we chose to examine whether a similar correlation existed between FE learning ability and acute arthritis pain-related behaviors and if this predictive value could be expanded to include both sexes.

Fear learning and FE were measured in 215 male and 190 female naïve rats (see Sections 2.1 and 2.5.1). During the fear learning session on day 1 of fear conditioning, rats showed minimal freezing behavior during the habituation phase under context A, indicating normal locomotor activity. All rats developed freezing responses after two pairings of CS (white noise, 80 dB, 4.5 kHz, 30 s) and US (0.7 mA foot shock, 2 s) (Figure 2A). During the fear training session on day 2, three groups emerged in both sexes based on differences in the time course and magnitude of declining freezing behavior in the absence of a foot shock (the US) (Figure 2B). For females, 36 rats (35.6%) exhibited a rapid (before 600 s; Phase I) decline in freezing to levels below 50% (per 30 s CS segment), reflecting strong FE learning ability (FE+), while 18 rats (17.8%) maintained freezing levels above 50% past 900 s (Phase III), indicating weak FE learning ability (FE−). The remaining 47 rats (46.5%) showed a decline to below 50% freezing levels between 600 and 900 s (Phase II) of the FE session and were classified as exhibiting "normal" FE learning ability (FE+/−). Males exhibited a different distribution of phenotypes, where 29 rats (19.8%) showed strong FE learning ability (FE+), 47 rats (30.7%) showed weak FE learning ability (FE−), and the remaining 77 rats (50.3%) showed normal FE learning ability (FE+/−) (Figure 2C). Female FE− rats showed a significantly higher percent freezing per 30 s CS segment than

those in the female FE+ group (*p* < 0.0001, F1,2080 = 512.8, two-way ANOVA; Bonferroni post hoc test results are shown in Figure 2B). Similarly, males in the FE− group showed a significantly higher percent freezing per 30 s CS segment than males in the FE+ group (*p* < 0.001, F1,2960 = 1372, two-way ANOVA; Bonferroni post hoc test results are shown in Figure 2B). Interestingly, FE+ males exhibited significantly lower percent freezing per 30 s CS segment than FE+ females (*p* < 0.01, F12,520 = 12.42, two-way ANOVA with Bonferroni post hoc tests) while FE− males showed significantly higher percent freezing per 30 s CS segment than FE− females (*p* < 0.0001, F12,520 = 22.75, two-way repeated-measures ANOVA with Bonferroni post hoc tests). Importantly, no differences in percent freezing were observed between the three groups for either sex during the habituation phases of the fear learning (Figure 2A) or the fear extinction (Figure 2B) sessions. the female FE+ group (*p* < 0.0001, F1,2080 = 512.8, two-way ANOVA; Bonferroni post hoc test results are shown in Figure 2B). Similarly, males in the FE− group showed a significantly higher percent freezing per 30 s CS segment than males in the FE+ group (*p* < 0.001, F1,2960 = 1372, two-way ANOVA; Bonferroni post hoc test results are shown in Figure 2B). Interestingly, FE+ males exhibited significantly lower percent freezing per 30 s CS segment than FE+ females (*p* < 0.01, F12,520 = 12.42, two-way ANOVA with Bonferroni post hoc tests) while FE− males showed significantly higher percent freezing per 30 s CS segment than FE− females (*p* < 0.0001, F12,520 = 22.75, two-way repeated-measures ANOVA with Bonferroni post hoc tests). Importantly, no differences in percent freezing were observed between the three groups for either sex during the habituation phases of the fear learning (Figure 2A) or the fear extinction (Figure 2B) sessions.

learning ability (FE+), 47 rats (30.7%) showed weak FE learning ability (FE−), and the remaining 77 rats (50.3%) showed normal FE learning ability (FE+/−) (Figure 2C). Female FE− rats showed a significantly higher percent freezing per 30 s CS segment than those in

*Brain Sci.* **2021**, *11*, x FOR PEER REVIEW 6 of 19

**Figure 2.** Inter-individual and sex differences in fear extinction learning ability in naïve female and male rats. Fear conditioning on Day 1 (**A**) and extinction (**B**) tests were conducted using two distinct context chambers. (**A**) Fear conditioning on Day 1—rats were habituated to context A followed by fear conditioning (2 CS-US pairs, see Section 2.5.1). The diagram illustrates the experimental protocol. Symbols in the line graph show freezing responses expressed in percent per 30 s segment during fear conditioning with 2 CS-US pairings. (**B**) Fear extinction learning on Day 2—rats were habituated to context B followed by extinction training (30 CSs, no US). The diagram illustrates the experimental protocol. Symbols in the line graph show freezing responses to tone (CS) expressed in percent per 30 s segment. (**C**) Bar histograms show the distribution of rats with strong (FE+), "normal" (FE+/−), and weak (FE-) fear extinction. The population (%) of FE+ was larger in female rats compared to male rats. For details, see the "Methods" and "Results" sections. CS: conditioned stimulus; US: unconditioned stimulus; ITI: intertone interval; FE: fear extinction. **Figure 2.** Inter-individual and sex differences in fear extinction learning ability in naïve female and male rats. Fear conditioning on Day 1 (**A**) and extinction (**B**) tests were conducted using two distinct context chambers. (**A**) Fear conditioning on Day 1—rats were habituated to context A followed by fear conditioning (2 CS-US pairs, see Section 2.5.1). The diagram illustrates the experimental protocol. Symbols in the line graph show freezing responses expressed in percent per 30 s segment during fear conditioning with 2 CS-US pairings. (**B**) Fear extinction learning on Day 2—rats were habituated to context B followed by extinction training (30 CSs, no US). The diagram illustrates the experimental protocol. Symbols in the line graph show freezing responses to tone (CS) expressed in percent per 30 s segment. (**C**) Bar histograms show the distribution of rats with strong (FE+), "normal" (FE+/−), and weak (FE−) fear extinction. The population (%) of FE+ was larger in female rats compared to male rats. For details, see the "Methods" and "Results" sections. CS: conditioned stimulus; US: unconditioned stimulus; ITI: intertone interval; FE: fear extinction.

#### *3.2. Inter-Individual and Sex Differences in Arthritis Pain-Related Behaviors of FE+ and FE*− *Rats* group, female FE+ and FE− rats spent significantly less time in the arena center compared

Next, we examined whether inter-individual and sex differences in FE learning ability would correspond with behavioral differences for males and females in an arthritis pain model (K/C arthritis, see Section 2.3) and/or in the untreated control condition. Male and female rats from the FE+ and FE− groups were selected for further behavioral testing and randomly assigned to either the K/C arthritis group or the untreated control group. Five weeks later (corresponding with an age-matched neuropathic pain group), arthritis was induced, and 6 h later, the following behavioral assays were performed: nocifensive reflexes (mechanosensitivity, Figure 3A) and ultrasonic and audible components of vocalizations (emotional responses, Figure 3B,C) evoked by mechanical compression of the knee joint, and the OFT (anxiety-like behavior, Figure 3D). to male FE+ and FE− rats, respectively (*p* < 0.0001, as shown in Figure 3D), suggesting higher baseline anxiety levels for females of both phenotypes. No significant differences in center duration were seen between females and males in the arthritis pain model for either phenotype. Importantly, no significant differences in locomotor activity were observed between the arthritis pain group and the untreated control group (*p* = 0.7327, Figure 3D), indicating that differences in anxiety-like behavior were not due to a reduction in spontaneous activity following arthritis induction. For the statistical analyses of OFT center duration in the four female experimental groups and the four male experimental groups, ANOVA with Bonferroni post hoc tests was used (female, F3,37 = 16.94; male, F3,31 = 72.79).

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**Figure 3.** Inter-individual and sex differences in arthritis pain-related behaviors of FE+ and FE− rats. (**A**) Mechanical thresholds tested in untreated control rats and arthritic rats (6 h post-induction) showed no significant differences between FE− (female, *n* = 7; male, *n* = 7) and FE+ (female, *n* = 7; male, *n* = 8) untreated rats or between FE− (female, *n* = 9; male, *n* = 7) and FE+ (female, *n* = 9; male, *n* = 8) arthritic rats, but arthritic FE− and FE+ rats had significantly lower withdrawal thresholds than their untreated controls. \*\*\*\* *p* < 0.0001, ANOVA with Bonferroni post hoc tests (see the "Results" section). (**B**,**C**) Duration (s) of ultrasonic and audible vocalizations, respectively, evoked by a brief (10 s) noxious (1500 g/30 mm2) mechanical compression of the knee. Significant differences in ultrasonic (but not audible) vocalizations were found between FE− (*n* = 9) and FE+ (*n* = 9) female arthritic rats but not between FE− (*n* = 7) and FE+ (*n* = 8) male arthritic rats or between untreated FE− (female, *n* = 11; male, *n* = 7) and FE+ (female, *n* = 13; male, *n* = 8) rats. For both sexes, arthritic rats had significantly increased vocalizations compared to their untreated controls. n.s.: non-significant; + *p* < 0.05; # *p* < 0.05; \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001, ANOVA with Bonferroni post hoc tests (see the "Results" section). (**D**) Center duration (s) in the OFT was significantly lower in arthritic FE− (female, *n* = 9; male, *n* = 7) and FE+ (female, *n* = 9; male, *n* = 13) rats compared to the untreated FE− (female, n = 8; male, n = 7) and FE+ (female, n = 15; male, n = 11) control rats. No differences were found between FE− and FE+ rats in the untreated control or arthritic groups for either sex. #### *p* < 0.0001; \*\*\*\* *p* < 0.0001, ANOVA with Bonferroni post hoc tests (see the "Results" section). Bar histograms show means ± SEM. FE: fear extinction; OFT: open field test. Asterisk (\*) indicates comparison to untreated group; plus sign (+) indicates comparison between phenotypes; pound sign (#) indicates comparison between sexes. **Figure 3.** Inter-individual and sex differences in arthritis pain-related behaviors of FE+ and FE− rats. (**A**) Mechanical thresholds tested in untreated control rats and arthritic rats (6 h post-induction) showed no significant differences between FE− (female, *n* = 7; male, *n* = 7) and FE+ (female, *n* = 7; male, *n* = 8) untreated rats or between FE− (female, *n* = 9; male, *n* = 7) and FE+ (female, *n* = 9; male, *n* = 8) arthritic rats, but arthritic FE− and FE+ rats had significantly lower withdrawal thresholds than their untreated controls. \*\*\*\* *p* < 0.0001, ANOVA with Bonferroni post hoc tests (see the "Results" section). (**B**,**C**) Duration (s) of ultrasonic and audible vocalizations, respectively, evoked by a brief (10 s) noxious (1500 g/30 mm<sup>2</sup> ) mechanical compression of the knee. Significant differences in ultrasonic (but not audible) vocalizations were found between FE− (*n* = 9) and FE+ (*n* = 9) female arthritic rats but not between FE− (*n* = 7) and FE+ (*n* = 8) male arthritic rats or between untreated FE− (female, *n* = 11; male, *n* = 7) and FE+ (female, *n* = 13; male, *n* = 8) rats. For both sexes, arthritic rats had significantly increased vocalizations compared to their untreated controls. n.s.: non-significant; <sup>+</sup> *p* < 0.05; # *p* < 0.05; \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001, ANOVA with Bonferroni post hoc tests (see the "Results" section). (**D**) Center duration (s) in the OFT was significantly lower in arthritic FE− (female, *n* = 9; male, *n* = 7) and FE+ (female, *n* = 9; male, *n* = 13) rats compared to the untreated FE− (female, *n* = 8; male, *n* = 7) and FE+ (female, *n* = 15; male, *n* = 11) control rats. No differences were found between FE− and FE+ rats in the untreated control or arthritic groups for either sex. #### *p* < 0.0001; \*\*\*\* *p* < 0.0001, ANOVA with Bonferroni post hoc tests (see the "Results" section). Bar histograms show means ± SEM. FE: fear extinction; OFT: open field test. Asterisk (\*) indicates comparison to untreated group; plus sign (+) indicates comparison between phenotypes; pound sign (#) indicates comparison between sexes.

> *3.3. Inter-Individual and Sex Differences in Neuropathic Pain-Related Behaviors of FE+ and FE− Rats*  As we previously reported that FE learning ability may serve as a predictor for neuropathic pain-related behaviors in male rats [42], we next sought to determine whether No significant differences in mechanical withdrawal thresholds were found between untreated FE+ rats (female, *n* = 7; male, *n* = 8) or untreated FE− rats (female, *n* = 7; male, *n* = 7) for either sex (Figure 3A). Similarly, no significant differences in mechanosensitivity

were found between FE+ rats (female, *n* = 9; male, *n* = 8) or FE− rats (female, *n* = 9; male, *n* = 7) in the arthritis pain model for either sex. However, mechanical withdrawal thresholds were significantly lower for arthritic female FE+ and FE− rats and for arthritic male FE+ and FE− rats compared to their untreated controls (*p* < 0.0001, as shown in Figure 3A), suggesting that both types of rats developed hypersensitivity in the pain model. No significant differences in mechanical withdrawal thresholds were found between female FE+ rats and male FE+ rats or between female FE− rats and male FE− rats for either the arthritis or untreated control groups. For the statistical analyses of mechanical withdrawal thresholds in the four female experimental groups and the four male experimental groups, ANOVA with Bonferroni post hoc tests was used (female, F3,28 = 53.09; male, F3,26 = 57.02).

For the ultrasonic and audible components of vocalizations (Figure 3B,C), no significant differences were found between untreated FE+ rats (female, *n* = 13; male, *n* = 8) and untreated FE− rats (female, *n* = 11; male, *n* = 7) for either sex. However, the total duration of vocalizations was significantly higher in female FE− rats (*n* = 9) than female FE+ rats (*n* = 9) in the arthritis pain model (*p* < 0.05, Figure 3B). No significant differences were found in the durations of audible components of vocalizations of these groups or in ultrasonic and audible components of vocalizations of male FE+ rats (*n* = 8) and male FE− rats (*n* = 7) in the arthritis group, though there was a non-significant trend (ultrasonic, *p* = 0.1988; audible, *p* = 0.1398). Total durations of ultrasonic and audible components of vocalizations were significantly increased for arthritic female FE+ and FE− rats and for arthritic male FE+ and FE− rats compared to their untreated controls (*p* < 0.05–0.0001, as shown in Figure 3B,C). Female FE− rats had significantly increased durations of ultrasonic but not audible components of vocalizations compared to male FE− rats (*p* < 0.05, as shown in Figure 3B) in the arthritis model. No differences were seen for durations of ultrasonic and audible components of vocalizations between female FE+ and male FE+ groups (untreated control or arthritis). Together, the data suggest that all groups developed emotional responses to arthritis pain, though it emerged most prominently for female FE− rats. For the statistical analyses of vocalization durations in the four female experimental groups and the four male experimental groups, ANOVA with Bonferroni post hoc tests was used (ultrasonic: female, F3,38 = 80.32, and male, F3,26 = 23.49; audible: female, F3,14 = 27.75, and male, F3,12 = 11.88).

In the OFT (Figure 3D), no significant difference in arena center duration was found between untreated FE+ rats (female, *n* = 15; male, *n* = 11) and untreated FE− rats (female, *n* = 8; male, *n* = 7) for either sex. Similarly, no significant differences in center duration were found between FE+ rats (female, *n* = 9; male, *n* = 13) and FE− rats (female, *n* = 9; male, *n* = 7) in the arthritis pain model for males or females. In the arthritis pain groups, female FE+ and FE− rats and male FE+ and FE− rats spent significantly less time in the center of the arena compared to their untreated controls (*p* < 0.0001, Figure 3D), suggesting all groups developed increased anxiety-like behavior. However, in the untreated control group, female FE+ and FE− rats spent significantly less time in the arena center compared to male FE+ and FE− rats, respectively (*p* < 0.0001, as shown in Figure 3D), suggesting higher baseline anxiety levels for females of both phenotypes. No significant differences in center duration were seen between females and males in the arthritis pain model for either phenotype. Importantly, no significant differences in locomotor activity were observed between the arthritis pain group and the untreated control group (*p* = 0.7327, Figure 3D), indicating that differences in anxiety-like behavior were not due to a reduction in spontaneous activity following arthritis induction. For the statistical analyses of OFT center duration in the four female experimental groups and the four male experimental groups, ANOVA with Bonferroni post hoc tests was used (female, F3,37 = 16.94; male, F3,31 = 72.79).
