*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 inter-individual differences in FE learning ability may also translate into behavioral differences for females in a neuropathic pain model (SNL, see Section 2.4) and/or in the sham control condition. Male and female FE+ and FE− rats were randomly assigned to the neuropathic pain group or sham group, and four weeks after SNL or sham surgery, the same behavioral assays were performed in these animals: nocifensive reflexes (Figure 4A) and ultrasonic and audible components of vocalizations (Figure 4B,C) evoked by mechanical compression of the hindpaw, and the OFT (Figure 4D). trol groups (*p* = 0.8120, Figure 4D) or between the sham control group and the untreated control group for the arthritis model (*p* = 0.4292, see Figure 3D), indicating that the observed differences in anxiety-like behavior were not due to a reduction in spontaneous activity following surgical procedures. For the statistical analyses of OFT center duration between the four female experimental groups and the four male experimental groups, ANOVA with Bonferroni post hoc tests was used (female, F3,78 = 6.119; male, F3,63 = 11.53).

and FE− rats (*p* < 0.0001, as shown in Figure 4D), suggesting higher anxiety levels for females at baseline, as also seen in naïve rats (see Figure 3D). Importantly, there were no significant differences in locomotor activity between the neuropathic pain and sham con-

*Brain Sci.* **2021**, *11*, x FOR PEER REVIEW 10 of 19

**Figure 4.** Inter-individual and sex differences in neuropathic pain-related behaviors of FE+ and FE− rats. (**A**) Mechanical thresholds tested in sham and chronic neuropathic SNL rats (4 weeks post-induction) showed no significant differences between FE− (female, *n* = 26; male, *n* = 32) and FE+ (female, *n* = 35; male, *n* = 40) sham rats or between FE− (female, *n* = 9; male, *n* = 14) and FE+ (female, *n* = 20; male, *n* = 17) SNL rats, but SNL FE− and FE+ rats had significantly lower withdrawal thresholds than their sham controls. \*\*\*\* *p* < 0.0001, ANOVA with Bonferroni post hoc tests (see the "Results" section). (**B**,**C**) Duration (s) of of ultrasonic and audible vocalizations, respectively, evoked by a brief (10 s) noxious (1500 g/6 mm2) mechanical compression of the affected hindpaw. Significant differences in ultrasonic and audible vocalizations were found between female FE− (*n* = 15) and FE+ (*n* = 20) SNL rats but not between male FE− (*n* = 15) and FE+ (*n* = 15) SNL rats or between FE− (female, *n* = 26; male, *n* = 32) and FE+ (female, *n* = 38; male, *n* = 41) sham rats. For both sexes, SNL rats had significantly increased vocalizations compared to their sham controls. n.s.: non-significant; + *p* < 0.05; # *p* < 0.05; ### *p* < 0.001; #### *p* < 0.0001; \*\*\*\* *p* < 0.0001, ANOVA with Bonferroni post hoc tests (see the "Results" section). (**D**) Center duration (s) in the OFT was significantly lower in FE− (female, *n* = 9; male, *n* = 13) and FE+ (female, *n* = 20; male, *n* = 20) SNL rats compared to their FE− (female, *n* = 19; male, *n* = 12) and FE+ (female, *n* = 33; male, *n* = 22) sham controls. No differences were found between FE− and FE+ rats in the sham or SNL groups for either sex. #### *p* < 0.0001; \* *p* < 0.05; \*\* *p* < 0.01; \*\*\*\* *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; SNL: spinal nerve ligation. Asterisk (\*) indicates comparison to untreated group; plus sign (+) indicates comparison between phenotypes; pound sign (#) indicates comparison between sexes. **Figure 4.** Inter-individual and sex differences in neuropathic pain-related behaviors of FE+ and FE− rats. (**A**) Mechanical thresholds tested in sham and chronic neuropathic SNL rats (4 weeks post-induction) showed no significant differences between FE− (female, *n* = 26; male, *n* = 32) and FE+ (female, *n* = 35; male, *n* = 40) sham rats or between FE− (female, *n* = 9; male, *n* = 14) and FE+ (female, *n* = 20; male, *n* = 17) SNL rats, but SNL FE− and FE+ rats had significantly lower withdrawal thresholds than their sham controls. \*\*\*\* *p* < 0.0001, ANOVA with Bonferroni post hoc tests (see the "Results" section). (**B**,**C**) Duration (s) of of ultrasonic and audible vocalizations, respectively, evoked by a brief (10 s) noxious (1500 g/6 mm<sup>2</sup> ) mechanical compression of the affected hindpaw. Significant differences in ultrasonic and audible vocalizations were found between female FE− (*n* = 15) and FE+ (*n* = 20) SNL rats but not between male FE− (*n* = 15) and FE+ (*n* = 15) SNL rats or between FE− (female, *n* = 26; male, *n* = 32) and FE+ (female, *n* = 38; male, *n* = 41) sham rats. For both sexes, SNL rats had significantly increased vocalizations compared to their sham controls. n.s.: non-significant; <sup>+</sup> *p* < 0.05; # *p* < 0.05; ### *p* < 0.001; #### *p* < 0.0001; \*\*\*\* *p* < 0.0001, ANOVA with Bonferroni post hoc tests (see the "Results" section). (**D**) Center duration (s) in the OFT was significantly lower in FE− (female, *n* = 9; male, *n* = 13) and FE+ (female, *n* = 20; male, *n* = 20) SNL rats compared to their FE− (female, *n* = 19; male, *n* = 12) and FE+ (female, *n* = 33; male, *n* = 22) sham controls. No differences were found between FE<sup>−</sup> and FE+ rats in the sham or SNL groups for either sex. #### *<sup>p</sup>* < 0.0001; \* *<sup>p</sup>* < 0.05; \*\* *<sup>p</sup>* < 0.01; \*\*\*\* *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; SNL: spinal nerve ligation. Asterisk (\*) indicates comparison to untreated group; plus sign (+) indicates comparison between phenotypes; pound sign (#) indicates comparison between sexes.

> Mechanical withdrawal thresholds showed no significant differences between sham FE+ rats (female, *n* = 35; male, *n* = 40) and sham FE− rats (female, *n* = 26; male, *n* = 32) for either sex (Figure 4A). Similarly, in the neuropathic pain model, there were no significant differences found in withdrawal thresholds between FE+ rats (female, *n* = 20; male, *n* = 17) and FE− rats (female, *n* = 9; male, *n* = 14) for either sex. Both female and male FE+ and FE− rats in the neuropathic pain model showed significantly lower mechanical thresholds compared to their sham controls (*p* < 0.0001, as shown in Figure 4A), suggesting both types of rats developed neuropathic hypersensitivity. No significant differences in mechanical withdrawal thresholds were found between female FE+ and male FE+ rats or between

female FE− and male FE− rats for either the neuropathic pain or sham control groups. For the statistical analyses of mechanical withdrawal thresholds between the four female experimental groups and the four male experimental groups, ANOVA with Bonferroni post hoc tests was used (female, F3,86 = 92.25; male, F3,99 = 46.40).

For ultrasonic and audible components of vocalizations (Figure 4B,C), no significant differences in duration were found between sham FE+ rats (female, *n* = 38; male, *n* = 41) and sham FE− rats (female, *n* = 26; male, *n* = 32) for either sex. However, the total durations of ultrasonic and audible components of vocalizations were significantly increased in female FE− rats (*n* = 15) compared to female FE+ rats (*n* = 20) in the neuropathic pain model (*p* < 0.05, as shown in Figure 4B,C). No significant differences in the durations of audible or ultrasonic components of vocalizations were found for male FE+ rats (*n* = 15) and male FE− rats (*n* = 15) in neuropathic pain. Both female and male FE+ and FE− rats in the neuropathic model had significantly increased durations of ultrasonic and audible components of vocalization compared to their sham controls (*p* < 0.0001, as shown in Figure 4B,C). In the neuropathic pain group, FE+ female rats had significantly greater durations of ultrasonic and audible components of vocalizations than FE+ male rats (ultrasonic: *p* < 0.0001, as shown in Figure 4B; audible: *p* < 0.05, as shown in Figure 4C) and FE− female rats had significantly greater durations of ultrasonic and audible components of vocalizations than FE− male rats (ultrasonic: *p* < 0.0001, as shown in Figure 4B; audible: *p* < 0.001, as shown in Figure 4C). Together, the data suggest that while all groups developed emotional responses to neuropathic pain, this occurred most prominently for FE− females. Individual examples of real-time waveforms and spectrogram recordings for phenotyped female and male SNL and sham control rats are shown in Figures 5 and 6. Though not reported in this study, our recordings (see Figures 5 and 6) suggest that similar differences between sexes and phenotypes may be observed with regard to the total number of vocalizations. Both the total duration [20,24] and the total number of calls [27,49] have been utilized as effective measures of behavioral responses in the context of pain. For the statistical analyses of ultrasonic and audible components of vocalizations between the four female experimental groups and the four male experimental groups, ANOVA with Bonferroni post hoc tests was used (ultrasonic: female, F3,89 = 125.9, and male, F3,96 = 25.90; audible: female, F3,66 = 177.9, and male, F3,56 = 66.50).

In the OFT (Figure 4D), no significant differences in arena center duration were found between sham FE+ rats (female, *n* = 33; male, *n* = 22) and sham FE− rats (female, *n* = 19; male, *n* = 12). Similarly, no differences in arena center duration were found between FE+ rats (female, *n* = 20; male, *n* = 20) and FE− rats (female, *n* = 9; male, *n* = 13) in the neuropathic pain model. Female FE+ (but not FE−) rats and male FE+ and FE− rats in the neuropathic pain group spent significantly less time in the center of the arena compared to their sham controls (*p* < 0.05–0.0001, as shown in Figure 4D). In the sham control group, female FE+ and FE− rats spent significantly less time in the center of the arena compared to male FE+ and FE− rats (*p* < 0.0001, as shown in Figure 4D), suggesting higher anxiety levels for females at baseline, as also seen in naïve rats (see Figure 3D). Importantly, there were no significant differences in locomotor activity between the neuropathic pain and sham control groups (*p* = 0.8120, Figure 4D) or between the sham control group and the untreated control group for the arthritis model (*p* = 0.4292, see Figure 3D), indicating that the observed differences in anxiety-like behavior were not due to a reduction in spontaneous activity following surgical procedures. For the statistical analyses of OFT center duration between the four female experimental groups and the four male experimental groups, ANOVA with Bonferroni post hoc tests was used (female, F3,78 = 6.119; male, F3,63 = 11.53).

zations.

**Figure 5.** Representative audible and ultrasonic vocalizations from phenotyped female rats in the SNL model of neuropathic pain. Original real-time waveform and spectrogram recordings of vocalizations evoked in response to brief (10 s) noxious (1500 g/6 mm2) mechanical stimulation of the affected hindpaw 4 weeks after induction of sham (**A**,**B**) or SNL (**C**,**D**) surgery in phenotyped female rats. For details, see Section 2.5.3. Mechanical stimuli were applied to the hindpaw in each recording period, as indicated by the highlighted yellow section of the waveform (upper panel, red arrow indicates initiation of noxious stimulus application); the total duration of the recording is 1 min. Boxes (events) in the spectrogram (lower panel) represent the presence of audible (blue; 20 Hz–16kHz) and ultrasonic (red; 25 ± 4 kHz) vocalizations during the 10 s application of mechanical stimuli. Female FE− sham rats (**A**) showed more vocalization events in response to noxious stimulus than female FE+ sham rats (**B**). Female FE− SNL rats (**C**) showed more vocalization events in response to noxious stimulus than female FE+ SNL rats (**D**). FE: fear extinction; SNL: spinal nerve ligation; USV: ultrasonic vocali-**Figure 5.** Representative audible and ultrasonic vocalizations from phenotyped female rats in the SNL model of neuropathic pain. Original real-time waveform and spectrogram recordings of vocalizations evoked in response to brief (10 s) noxious (1500 g/6 mm<sup>2</sup> ) mechanical stimulation of the affected hindpaw 4 weeks after induction of sham (**A**,**B**) or SNL (**C**,**D**) surgery in phenotyped female rats. For details, see Section 2.5.3. Mechanical stimuli were applied to the hindpaw in each recording period, as indicated by the highlighted yellow section of the waveform (upper panel, red arrow indicates initiation of noxious stimulus application); the total duration of the recording is 1 min. Boxes (events) in the spectrogram (lower panel) represent the presence of audible (blue; 20 Hz–16 kHz) and ultrasonic (red; 25 ± 4 kHz) vocalizations during the 10 s application of mechanical stimuli. Female FE− sham rats (**A**) showed more vocalization events in response to noxious stimulus than female FE+ sham rats (**B**). Female FE− SNL rats (**C**) showed more vocalization events in response to noxious stimulus than female FE+ SNL rats (**D**). FE: fear extinction; SNL: spinal nerve ligation; USV: ultrasonic vocalizations.

**Figure 6.** Representative audible and ultrasonic vocalizations from phenotyped male rats in the SNL model of neuropathic pain. Original real-time waveform and spectrogram recordings of vocalizations evoked in response to brief (10 s) noxious (1500g/6mm2) mechanical stimulation of the affected hindpaw 4 weeks after induction of sham (**A**,**B**) or SNL (**C**,**D**) surgery in phenotyped male rats. For details, see Section 2.5.3. Mechanical stimuli were applied to the hindpaw in each recording period, as indicated by the highlighted yellow section of the waveform (upper panel, red arrow indicates initiation of noxious stimulus application); the total duration of the recording is 1 min. Boxes (events) in the spectrogram (lower panel) represent the presence of audible (blue; 20 Hz–16kHz) and ultrasonic (red; 25 ± 4 kHz) vocalizations during the 10 s application of mechanical stimuli. Male FE− sham rats (**A**) showed more vocalization events in response to noxious stimulus than male FE+ sham rats (**B**). Male FE− SNL rats (**C**) showed more vocalization events in response to noxious stimulus than male FE+ SNL rats (**D**). FE: fear extinction; SNL: spinal nerve ligation; USV: ultrasonic vocalizations. **Figure 6.** Representative audible and ultrasonic vocalizations from phenotyped male rats in the SNL model of neuropathic pain. Original real-time waveform and spectrogram recordings of vocalizations evoked in response to brief (10 s) noxious (1500 g/6 mm<sup>2</sup> ) mechanical stimulation of the affected hindpaw 4 weeks after induction of sham (**A**,**B**) or SNL (**C**,**D**) surgery in phenotyped male rats. For details, see Section 2.5.3. Mechanical stimuli were applied to the hindpaw in each recording period, as indicated by the highlighted yellow section of the waveform (upper panel, red arrow indicates initiation of noxious stimulus application); the total duration of the recording is 1 min. Boxes (events) in the spectrogram (lower panel) represent the presence of audible (blue; 20 Hz–16 kHz) and ultrasonic (red; 25 ± 4 kHz) vocalizations during the 10 s application of mechanical stimuli. Male FE− sham rats (**A**) showed more vocalization events in response to noxious stimulus than male FE+ sham rats (**B**). Male FE− SNL rats (**C**) showed more vocalization events in response to noxious stimulus than male FE+ SNL rats (**D**). FE: fear extinction; SNL: spinal nerve ligation; USV: ultrasonic vocalizations.

#### **4. Discussion 4. Discussion**

This study explored the predictive value of FE learning ability in sensory and affective pain-related behaviors for male and female animals in an acute arthritis and a chronic neuropathic pain model. We previously showed a positive correlation between FE learning ability and neuropathic pain behaviors in adult male rats [42], but it is unclear if these are also found in acute pain conditions and whether female rats exhibit a similar association. The key novelties of this study are the identification of distinct behavioral phenotypes based on FE learning ability for both sexes, with vocalizations being the most effective indicators, and that these behavioral phenotypes show striking differences between male and female rats in both pain models. This study explored the predictive value of FE learning ability in sensory and affective pain-related behaviors for male and female animals in an acute arthritis and a chronic neuropathic pain model. We previously showed a positive correlation between FE learning ability and neuropathic pain behaviors in adult male rats [42], but it is unclear if these are also found in acute pain conditions and whether female rats exhibit a similar association. The key novelties of this study are the identification of distinct behavioral phenotypes based on FE learning ability for both sexes, with vocalizations being the most effective indicators, and that these behavioral phenotypes show striking differences between male and female rats in both pain models.

Fear learning and extinction assays were selected as approaches to identifying interindividual differences in pain-related behaviors because these are well-established models for correlating animal behavior with neural structure and function [15]. Previous studies from our lab and others have reported the separation of fast and slow recovery phenotypes based on freezing levels during FE that correlate with differences in anxiety-like behavior [16,42,50–52]. At the clinical level, inter-individual differences in fear response modulation and generalization have been linked to increased vulnerability in the development of anxiety disorders and post-traumatic stress disorder (PTSD) [15,17,53–55], and patients with anxiety disorders, PTSD, and obsessive-compulsive disorder (OCD) have exhibited delayed and/or reduced FE or extinction recall [14,56–59]. A previous epidemiological study reported that most individuals who experience trauma recover, with only a subset going on to develop a psychopathology such as depression or anxiety [60]. Similarly, chronic widespread pain develops in only 10% of the population [61]. However, a major goal of preclinical research is to provide insights into neural processes and behaviors that can predict susceptibility versus resistance to a disorder. This requires the study of neural variability patterns that differ from the central tendency. Thus, we chose to focus on (representatives of) the groups at the two ends of the spectrum (weak FE learning ability (FE−), considered to be "susceptible" rats, versus strong FE learning ability (FE+), considered to be "resistant" rats) within this study instead of including the larger, "normal" FE+/− group.

Little has been studied with regard to sex differences in classic fear conditioning and extinction models. This is an important knowledge gap, as females have twice the lifetime rates of depression and anxiety disorders [62], and human imaging studies revealed structural and functional sex differences in anxiety-relevant brain regions [63]. One preclinical study found that, while fast and slow extinction phenotypes could be identified for both sexes, there were no observable differences between males and females in freezing levels during fear conditioning or extinction [64]. Others have reported impairments in FE recall for female rats when compared to male rats [65,66]. However, several studies have reported that females have greater FE rates when compared to males [67–70]. A recent review suggested that sex hormones may play an important role in conditioned FE, as estrous cycle influences may affect female FE mechanisms [71]. The results of this study are consistent with those from the literature that showed gonadectomized males spent a greater amount of time freezing than gonadectomized females, a pattern that was not affected by estradiol administration [68]. Another study suggested that endogenous estrogen did not affect FE behavior in female rats or naturally cycling women [72]. Therefore, it is unlikely that the estrous cycle significantly affected the freezing levels of females in our study.

Inter-individual differences have been well-documented for pain and pain modulation [5]. Neurobiological mechanisms, including emotional network plasticity, may link pain and fear [11]. This relationship has been explored with regard to the corticolimbic system [73,74], and in particular, the amygdala, a limbic structure that has emerged as a key player in both fear and anxiety networks [75–78] and in the emotional-affective dimensions of pain and pain modulation [8,10,79]. Corticolimbic characteristics involving the amygdala determine the risk of chronic pain and mediate the effects of depression and negative affect on chronic pain [80]. Human studies have investigated the role of the amygdala in pain and fear interactions [73,81], and the amygdala has been implicated in fear-conditioned analgesia in a preclinical setting [82–84]. The amygdala has been implicated in pain-like behaviors [85–87], anxiety-like behaviors [88–90], and in fear learning [12,91]. Pain-related neuroplastic changes lead to hyperexcitability in amygdala output neurons [10], driving pain behaviors in both acute [92,93] and chronic [29,94] pain models. Sex differences with regard to pain conditions have long been recognized, with females greatly outnumbering males as chronic pain patients [6]. However, sex differences in pain-related amygdala neuroplasticity are largely unknown, though one clinical study reported sex differences in resting-state amygdala subnuclei connectivity patterns as a potential explanation for the increased prevalence of conditions of negative affect in women [95]. Even less has been

explored about inter-individual and sex differences in fear learning and FE with regard to pain and pain modulation, though a clinical study reported sex differences in pain-related fear conditioning [96]. Ultrasonic vocalizations were previously associated with increased neuronal activity in brain regions regulating fear and anxiety, including the amygdala [97], and have been demonstrated to be an effective indicator of emotional status in pain models [20,21,27]. As ultrasonic vocalizations demonstrated the most striking inter-individual and sex differences in pain-related behaviors for both an acute and a chronic model, insight into potential sexual dimorphisms of pain-related amygdala neuroplasticity is warranted.

The intricate relationship between pain modulation and fear neurocircuitry and mechanisms, particularly in relation to potential discrepancies regarding sex differences, led us to test the hypothesis that FE learning ability can predict pain-related behaviors in both acute (arthritis) and chronic neuropathic (SNL) models of pain, and that these behaviors may differ between males and females. In the present study, distinct behavioral phenotypes differed according to sex in their FE but not fear learning ability. There were no differences at baseline between mechanosensitivity (spinal reflex thresholds) and emotional-affective responses (vocalizations), but females exhibited increased baseline anxiety-like behavior (OFT) compared to males in both the untreated and sham-treated control groups (see Figures 3D and 4D). This confirms findings from the literature that males spent the same or increased time in the center of the OFT compared to females at baseline [3,98], though one study found no sex difference in OFT anxiety-like behavior in a chronic spinal nerve transection pain model [99]. FE+ and FE− phenotypes showed differences in the magnitude of emotional-effective responses not only in the neuropathic pain model, as we previously reported [42], but also in the arthritis pain model (see Figure 3B,C and Figure 4B,C). Additionally, females exhibited significantly increased audible and ultrasonic components of vocalizations compared to males in both of the tested pain models. To the best of our knowledge, sex differences in pain-related vocalizations in the context of FE learning have not been reported. One preclinical study found that male rats vocalized more than female rats despite females exhibiting lower freezing levels during FE, although this effect was strain-specific and did not include any pain models [100]. The novelty here is the identification of sex-specific differences in behavioral phenotypes, which corresponds to sexual dimorphisms in pain-related vocalizations regardless of pain model.

Sonic vocalizations, if emitted with large force and volume, may produce overtones that reach into the ultrasonic frequency range. A note of consideration in the present study is that the ultrasonic components (harmonics) of audible vocalizations presented here cannot be regarded as true ultrasonic aversive vocalizations as rats cannot emit sonic and ultrasonic calls at the same time. However, our results show that harmonic components of vigorous audible vocalizations showed an interesting harmonic spectrum, possibly with additional overtones. Because some of the overtones may depart from the whole multiples of the fundamental frequency, the harmonics and overtones show reinforcement at higher frequencies, creating ultrasonic components of the audible calls that are clearly visible in the spectrograms. Ultrasonic components of vocalizations are of long duration, consistent with the duration of audible calls. Simultaneous audible and ultrasonic vocalization components were demonstrated in response to an acute painful stimulus (tail snip) [101]. Ultrasonic harmonics that were previously reported demonstrated a different duration and lower frequency than presented here [102]. Though the emission of 22 kHz ultrasonic vocalizations has been reported to occur after a significant delay [103–105], in this study, both audible and ultrasonic components were evoked by a continuously present mechanical stimulus for 10 s as opposed to the brief electrical stimuli used in other studies. Repeated vocalizations may be triggered by the continuous noxious stimulus, and thus, latency assessment is not possible with this approach. The use of audible vocalizations in both the audible and ultrasonic ranges, particularly in correlation with other behavioral measures, is a useful measure of pain levels and emotional responses to pain.

The current study provides the rationale for the inter-individual- and sex-specific analysis of synaptic and cellular mechanisms within the amygdala. Future research may address neuroplastic differences between males and females in the context of pain and fear learning, potentially providing insight into the increased prevalence of anxiety, PTSD, and pain in female patients and supporting patient-specific therapeutic strategies for these disorders [15].

#### **5. Conclusions**

The data may suggest sexual dimorphisms in FE learning ability that have a predictive value for pain-related behavioral changes, particularly among emotional-affective pain aspects, in both an acute and a chronic pain model. Rats with weak FE learning ability showed an increased magnitude of both arthritic- and neuropathic-pain related affective rather than sensory behaviors, with females demonstrating greater inter-individual differences in affective pain behaviors than males. Vocalizations are strong indicators of inter-individual and sex differences in pain models, particularly in chronic neuropathic pain, whereas no such differences were found for mechanosensitivity, and anxiety-like behaviors showed only baseline differences. The increased correlation between FE learning ability and affective pain-related behaviors in female compared to male rats may be facilitated by amygdala pain mechanisms, though further investigation into sex-specific synaptic and cellular neurobiological mechanisms is warranted.

**Author Contributions:** Conceptualization, V.N., P.P. and G.J.; methodology, V.N., P.P. and G.J.; validation, P.P., G.J., R.J. and Z.G.; formal analysis, P.P., G.J., R.J., Z.G. and V.N.; investigation, P.P., G.J., R.J. and Z.G.; resources, V.N.; writing—original draft preparation, P.P.; writing—review and editing, V.N. and P.P.; visualization, P.P. and V.N.; supervision, V.N. and G.J.; project administration, V.N. and G.J.; funding acquisition, V.N. and G.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Institutes of Health, grant number R01 NS038261, R01 NS106902, R01NS118731, R01 NS120395, and R01 NS109255.

**Institutional Review Board Statement:** Experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC; protocol #14006, currently approved through 20 June 2022) at Texas Tech University Health Sciences Center.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data generated or analyzed during this study are included in this published article. Data files used for this manuscript are available via a direct and reasonable request to the corresponding author and approval from Texas Tech University Health Sciences Center (TTUHSC).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


MDPI St. Alban-Anlage 66 4052 Basel Switzerland Tel. +41 61 683 77 34 Fax +41 61 302 89 18 www.mdpi.com

*Brain Sciences* Editorial Office E-mail: brainsci@mdpi.com www.mdpi.com/journal/brainsci

MDPI St. Alban-Anlage 66 4052 Basel Switzerland

Tel: +41 61 683 77 34 Fax: +41 61 302 89 18

www.mdpi.com ISBN 978-3-0365-3284-4