We thank the Animal Resources Center (ARC) husbandry staff at the Health Discovery Building for excellent animal care, and Dr. Laura Fonken for helpful critique and feedback on the manuscript. Partial support was provided by University of Texas at Austin start-up funds (ADG), the Wings for Life Foundation (ADG) and Mission Connect, a program of the TIRR Foundation (ADG)..

Spinal cord injury (SCI) predisposes individuals to anxiety and chronic pain. Anxiety- and pain-like behavior after SCI can be tested in rodents, yet commonly used tests assess one variable and may not replicate effects of SCI or sex differences seen in humans. Thus, novel preclinical tests should be optimized to better evaluate behaviors relating to anxiety and pain. Here, we use our newly developed conflict test - the Thermal Increments Dark-Light (TIDAL) test - to explore how SCI affects anxiety- vs. pain-like behavior, and whether sex affects post-SCI behavior. The TIDAL conflict test consists of two plates connected by a walkway; one plate remains illuminated and at an isothermic temperature, whereas the other plate is dark but is heated incrementally to aversive temperatures. Control mice are tested with both plates illuminated (thermal place preference). Female and male mice received moderate T9 contusion SCI or remained uninjured. At 7 days post-operative (dpo), mice with SCI increased dark plate preference throughout the TIDAL conflict test compared to uninjured mice. SCI increased dark plate preference for both sexes, although female (vs. male) mice remained on the heated-dark plate to higher temperatures. Mice with SCI that repeated TIDAL at 7 and 21 dpo showed reduced preference for the dark-heated plate at 21 dpo. Overall, in female and male mice, SCI enhances the salience of anxiety (vs. heat sensitivity). The TIDAL conflict test meets a need for preclinical anxiety- and pain-related tests that recapitulate the human condition; thus, future rodent behavioral studies should incorporate TIDAL or other conflict tests to help understand and treat neurologic disorders.

Anxiety and chronic pain afflict hundreds of millions worldwide. Both anxiety and pain are more prevalent in females (vs. males). Unfortunately, robust sex differences in human anxiety are not recapitulated in rodent tests, and results from rodent pain studies frequently fail to translate clinically. Therefore, there is a need to develop tests that reflect the differential salience of anxiety or pain-related stimuli between the sexes. Accordingly, here we introduce the Thermal Increments Dark-Light (TIDAL) conflict test. The TIDAL test places an anxiety-relevant stimulus (dark vs. illuminated chamber) in conflict with a heat-related stimulus (incrementally heated vs. isothermic chamber); mice freely explore the dark-heating and illuminated-isothermic chambers. Here, we aim to determine whether the TIDAL conflict test reveals in mice underappreciated sex differences in anxiety and/or heat sensitivity. We establish in three distinct experiments that females on the TIDAL conflict test persist substantially longer on the dark-heated plate, suggesting that females (vs. males) exhibit elevated anxiety-like behavior. Mice more strongly prefer the dark plate on the TIDAL conflict test compared to control thermal place preference with both chambers illuminated. We also reveal that mice exposed to TIDAL for a second session exhibit signs of learning. Therefore, our new TIDAL conflict test reliably unmasks the relative salience of anxiety (vs. heat sensitivity): mice that are female exhibit robust anxiety-like behaviors not consistently observed in classical tests. Future studies should incorporate TIDAL and other conflict tests to better understand rodent behavior and to identify mechanisms underlying anxiety and pain.

Spinal cord injury (SCI) in humans frequently causes intractable chronic pain. Females are susceptible to worsened pain compared to males, and females may show higher pain prevalence after SCI in humans. Despite this difference in clinical prevalence of SCI pain, few preclinical studies have systematically studied in rodents sex differences in SCI-elicited pain symptoms. Here, we leverage data from a large cohort of mice to test whether contusion SCI consistently causes pain symptoms in mice, and to establish whether female (vs. male) mice display heightened hypersensitivity after SCI. Mechanical and heat sensory thresholds were assessed using the von Frey test and Hargreaves test, respectively. In an initial experiment, female mice receiving moderate 60 kDyn SCI or moderate-to-severe 75 kDyn SCI both exhibited mechanical and heat pain symptoms compared to sham controls. 75 kDyn SCI caused excess motor deficits that confounded defining pain sensitivity at acute times, so the moderate SCI force was used for subsequent experiments. Next, adult female and male C57BL6/J mice received sham surgery or T9 contusion SCI. Comparing female to male mice after SCI, we reveal that mice of both sexes displayed mechanical and heat hypersensitivity compared to sham controls, from acute-to-chronic post-injury times. Females had amplified SCI-elicited hypersensitivity compared to males. Our data suggest that thoracic contusion SCI elicits consistent and persistent pain symptoms, which are more intense in female vs. male mice. These results have important implications for uncovering sex-specific mechanisms and therapeutic targets to ameliorate neuropathic pain after SCI.