The Analgesic Effect of Neuropeptide S (NPS) in Alcohol-Dependent Male and Female Rats

Abstract

Introduction:

Alcohol dependence (AD) includes tolerance to alcohol’s analgesic effects and increased pain sensitivity during withdrawal (i.e., hyperalgesia). Further, the reciprocal relationship between alcohol use and pain sensitivity is hypothesized to contribute to the maintenance of AD through negative reinforcement (i.e., self-medication). The neuropeptide S (NPS) system, known to regulate stress, arousal, and pain pathways, may offer a therapeutic target to ameliorate AD-induced hyperalgesia. Given previous reports on the antinociceptive properties of NPS, we hypothesized that central administration of NPS could attenuate the heightened pain sensitivity observed in AD rats.

Methods:

Male (n=11) and female (n=7) Wistar rats were surgically implanted with an intracerebroventricular (ICV) cannula and assigned to either the AD or alcohol-naïve control group, matched in terms of their baseline thermal nociceptive thresholds. Pain sensitivity was tracked weekly using thermal (Hargreaves) and mechanical (robotic Von Frey, also known as the dynamic plantar aesthesiometer) assays to establish AD-induced hyperalgesia. After stable hyperalgesia emerged, rats received ICV NPS administrations (0, 0.1, or 1 nmol in 5 μL saline) 5 minutes prior to pain testing (Experiment 1: Hargreaves; Experiment 2: robotic Von Frey). In a follow-up experiment, three Von Frey methods (electronic, robotic, and manual) were compared to assess their efficacy in detecting AD-induced mechanical hyperalgesia.

Results:

AD male and female rats developed significant hyperalgesia across both pain modalities. Central administration of NPS produced robust analgesia in a dose-dependent manner in both AD and alcohol-naïve control rats. No sex differences were observed in baseline nociception, AD-induced hyperalgesia, or NPS-induced analgesia. All three mechanical assays reliably detected AD-induced mechanical hyperalgesia, and the importance of adequate habituation procedures is discussed.

Conclusion:

These findings indicate that NPS exerts a robust analgesic effect in male and female rats. This effect appears independent of ethanol-exposure history but produces sufficient analgesia to alleviate pain sensitivity in hyperalgesic AD rats to/beyond the level of sensitivity in vehicle treated, alcohol-naïve controls. The NPS system may therefore represent a promising, non-addictive target for treating pain-related symptoms in alcohol dependence.

Introduction:

Alcohol use disorder (AUD) is a leading cause of preventable death in the United States, that contributes to over 178,000 deaths annually according to the most recently available estimates(Centers for Disease Control and Prevention, 2024a, 2024b). Of those deaths, about two-thirds (117,000) were attributable to chronic conditions such as alcohol dependence (AD), that increase the risk of developing liver disease, heart disease, and other comorbidities(Centers for Disease Control and Prevention, 2024a, 2024b). AD is characterized by escalated and compulsive alcohol drinking, and it is hypothesized that exacerbated brain stress signaling contributes to the enhanced motivation to consume alcohol in AD (i.e., consuming alcohol for relief from stress and negative emotion)(Herman et al., 2013; Koob, 2021; Pantazis et al., 2021; Roberto et al., 2021; Sinha, 2012; Tunstall et al., 2017; Tunstall et al., 2020; Varodayan et al., 2025).

Studies suggest that symptoms of AD include enhanced pain sensitivity in alcohol withdrawal (i.e., hyperalgesia), that is hypothesized to contribute to the motivation to drink alcohol(Alexander et al., 2023; Borgonetti et al., 2023; Brandner et al., 2023; Hall et al., 2024; Koob & Vendruscolo, 2025; LeBlanc et al., 2015; Maleki et al., 2019; Marendes et al., 2025; Wade et al., 2014). Recently there has been great interest in developing treatments that could target hyperalgesia in alcohol dependence as component of the AUD phenotype(Koob & Vendruscolo, 2025). Pain is a debilitating condition that not only can impair physical functioning, but can also disrupt emotional well-being, cognitive performance, and overall quality of life(Cohen et al., 2021; Poisbeau & Salvat, 2025a, 2025b; Woolf, 2010). During 2021 an estimated 51.6 million (20.9%) of U.S. adults experienced chronic pain, with important disparities in the prevalence of the populations affected(Duca et al., 2022; Rikard et al., 2023). Examples include veterans, individuals living in poverty, and those with pre-existing chronic health conditions, which are populations that are also prevalent in those with AD(Duca et al., 2022; Rikard et al., 2023). Individuals with chronic pain are up to twice as likely as the general population to meet criteria for AUD, with 75% of those with AUD reporting moderate to severe pain(Zale, 2021). Studies on human subjects commonly report that relief of pain is a reason for use of opioids and alcohol(Ditre et al., 2023; Edwards et al., 2020; Egli et al., 2012; Koob, 2020, 2021; LeBlanc et al., 2015; McGinn et al., 2021; Wade et al., 2014). More than half of patients with AD experience increased persistent pain that includes alcoholic neuropathy, with many patients reporting that they seek alcohol to relieve pain (Behl et al., 2021; Mellion et al., 2011).

Unfortunately, the self-medication of pain states using alcohol can worsen the development of hyperalgesia in alcohol withdrawal, and heightened pain sensitivity is a well described symptom of alcohol dependence and withdrawal in humans and rodents alike(Alexander et al., 2023; Brandner et al., 2023; Hall et al., 2024; Koob & Vendruscolo, 2025; LeBlanc et al., 2015; Maleki et al., 2019; Marendes et al., 2025; Wade et al., 2014).This process is reminiscent of, and shares some neurobiological overlap with, the —more extensively studied— self-medication of pain using opioids, and the development of hyperalgesia in opioid dependence and withdrawal in rats(Alvarez-Bagnarol et al., 2023; Marchette et al., 2021; Monroe & Radke, 2023; Rieb et al., 2018; Vendruscolo et al., 2018; Wade et al., 2014) and mice(Moussawi et al., 2020). This maladaptive cycle not only contributes to the motivation to consume more alcohol (and/or opioids), but further complicates treatment outcomes, raising the need for integrated, dual-focused treatment approaches that simultaneously address pain and AD symptoms (Edwards et al., 2020; Egli et al., 2012; Koob & Vendruscolo, 2025; LeBlanc et al., 2015; Robins et al., 2019). Preclinical efforts have begun to model AD-induced hyperalgesia and elucidate its underlying neurobiology(Avegno et al., 2018; Brandner et al., 2023; Gilpin et al., 2021; Marendes et al., 2025; Secci et al., 2024). Given the reliability of rodent pain measures (i.e., Hargreaves and Von Frey)(Cheah et al., 2017; Deuis et al., 2017; Modi et al., 2023) and the direct translational relevance for pain mechanisms in humans(Edwards et al., 2020; Koob & Vendruscolo, 2025), rodent models represent a great opportunity to test the role of neurobiological targets for their putative therapeutic effect in AD-induced hyperalgesia.

In the present study, we sought to examine the anti-nociceptive effects of neuropeptide S (NPS), which, based on the existing literature, we hypothesize could ameliorate the hyperalgesia observed in AD(Cannella et al., 2013; Enquist et al., 2012; Grund et al., 2017; Kushikata et al., 2021; Lee et al., 2020; Li et al., 2009; Reinscheid et al., 2005; Ren et al., 2013; Xu et al., 2004). NPS is a 20 amino acid neuropeptide that is produced almost entirely in a few brainstem subnuclei in humans and rodents(Xu et al., 2004; Xu et al., 2007) and has been shown to have a unique profile of action that includes both antinociceptive(Cannella et al., 2022; Kushikata et al., 2021; Lee et al., 2020; Marendes et al., 2025) and anti-stress/anxiolytic effects(Cannella et al., 2022; Kushikata et al., 2021; Lee et al., 2020; Pape et al., 2010; Reinscheid et al., 2005; Tillmann et al., 2019), while in tandem increasing locomotion/arousal(Ensho et al., 2017; Li et al., 2015; Reinscheid & Ruzza, 2021). Research in rats suggests the NPS/NPS-receptor system is dysregulated in AD, as in situ hybridization during alcohol withdrawal revealed compensatory increased NPS receptor mRNA expression in brain regions involved in pain such as the lateral hypothalamus, paraventricular nucleus, and basolateral and central amygdala(Ruggeri et al., 2010). Similar findings have also been reported in studies of opioid dependent rats, with increases of NPS receptor MRA expression during morphine withdrawal(Ghazal et al., 2013). These observations point to a potential link between the NPS system and pain-related processes of alcohol withdrawal. Thus, based on the available body of literature we hypothesized that central administration of NPS in a rat model of AD has the potential to normalize this dysregulated state and ameliorate AD-induced hyperalgesia.

This study also sought to determine potential sex differences in any effects observed, given that males and females may experience sensitized pain from alcohol exposure differently(Alexander et al., 2023; Brandner et al., 2023; Li et al., 2019; Marendes et al., 2025; Rebecca M Craft, 2004). For example, female rats have been shown to be more sensitive than males in their mechanical nociceptive threshold(Hendrich et al., 2012), whereas male rats have been shown to be more sensitive than females in their thermal nociceptive threshold(Bourgeois et al., 2022). Further, we anticipated potential sex differences in the effects of NPS administration, as prior studies report sex differences in endogenous NPS release following stress exposure, where high stress reactivity in female mice compared to males was associated with relatively high and low release levels of NPS, respectively(Bengoetxea et al., 2021). Our previous work was not designed to compare male and female nociception directly(Marendes et al., 2025), and so the present study also sought to identify any sex differences in the methods of pain testing and the hypothesized analgesic effect of NPS.

Our primary hypothesis was that the analgesic effect of NPS has the potential to ameliorate the hyperalgesia that develops in alcohol dependent rats. To test this hypothesis, we made rats alcohol dependent using chronic intermittent ethanol vapor (CIEV) exposure and compared them with an alcohol-naïve control group. Then we monitored the pain sensitivity in both groups across several weeks on both thermal (Hargreaves) and mechanical (Von Frey) pain assays. Finally, we administered ICV NPS in all rats prior to tests of both pain modalities to examine the ability of NPS to reverse AD-induced hyperalgesia. In addition, to determine the reliability of the AD-induced mechanical hyperalgesia phenotype, we directly compared three methods currently employed commonly in the pain field to assay mechanical nociception in rodents: electronic-handheld Von Frey; robotic Von Frey (also known as the dynamic plantar aesthesiometer); and the manual Von Frey.

Materials and Methods:

Experimental procedures were conducted using methods similar to those reported previously(Marendes et al., 2025), with minor modifications detailed here.

Animals and Housing:

Male (n= 11) and female (n= 7) Wistar rats arrived at the animal facility at 5 weeks of age and weighing 76–100 grams (Charles River Laboratories International, Inc., Wilmington, MA) and were allowed at least 4 weeks to adapt to the animal colony before surgery. Throughout the experiment, all rats were group-housed and given ad libitum food and water while in the home cage, and housed on a 12:12 reversed light:dark cycle (lights on 6pm, off 6am). Animal colony rooms were temperature (approximately 21 C) and humidity (approximately 31% relative humidity) controlled. All behavioral studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Tennessee Health Science Center and all procedures were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, 8th edition.

Surgery:

At 9 weeks of age, all rats received intracerebroventricular (ICV) cannulation surgery performed under sterile conditions, to allow direct delivery of compounds into the lateral ventricle. Animals were anesthetized using [isoflurane (1.5–2.5%) in oxygen] and placed into a stereotaxic frame (RWD Life Sciences, Shenzhen, China) with non-rupture ear bars to stabilize the head. They were implanted with a 26-gauge ICV guide cannula, cut 5 mm below the pedestal (RWD Life Sciences, Shenzhen, China). The cannula was targeted to the left lateral ventricle (stereotaxic coordinates, with upper incisor bar set to achieve “flat skull” orientation: A/P = −0.72 mm, M/L = 1.70 mm, and D/V = −3.60 mm from skull surface). The cannula was fixed to the skull with dental cement anchored by 4 stainless steel screws. Once the cement was fully dry, the cannula was occluded by a custom stylet prepared with stainless steel, 0.012″-diameter spring-back wire, flush-cut to extend 1 mm beyond the end of the implanted guide cannula, with the cannula-stylet assembly protected by a threaded metal cap. Correct cannula placement was verified after recovery from surgery by observing the ICV free-flow of saline through the infusion line by gravity (see “Injections”) and again at the end of the experiment by dye injection (2% Evans blue).

Animals received subcutaneous injections of an NSAID (e.g., meloxicam 1 mg/kg) to reduce pain and an antibiotic (e.g., gentamicin 3.2 mg/kg) to reduce bacterial infection post-operatively. Animals were allowed to recover in a heated chamber until fully ambulatory and monitored daily for at least 3 days post-surgery. All rats were given 5 weeks to recover from surgery before pain testing.

Injections:

ICV infusions were made using a 30-gauge injector cut to project 1 mm beyond the end of the implanted guide cannula. The injector was connected by a PE-10 tubing infusion line to a 10 μL Hamilton^®, Model 1701 LT syringe, terminated with a 27-gauge blunt needle tip, allowing manual infusion of precise infusion volumes. All “dead volume” in the infusion system including the Hamilton syringe was filled with saline, separated from the <10μl drug treatment solution at the injector-end of the infusion setup by a single, small (<5mm length) air bubble that allowed the experimenter to mark and visually confirm the steady flow of solution during manual injections.

Drugs and Pretreatment Times:

Neuropeptide S (MedChemExpress, Monmouth Junction, NJ) was dissolved in 0.9% saline for ICV injections. ICV NPS treatments were administered 5 min prior to the Hargreaves or robotic Von Frey tests in order for the beginning of testing to coincided with estimated peak plasma and brain concentrations of NPS in the rat (see Supplement File 1 for a time course of NPS’s enhancement of locomotion, assessed in a separate cohort of rats;(Cannella et al., 2016; Rizzi et al., 2008; Xu et al., 2004)), so that testing would be conduct during a period of elevated peptide concentrations (note: pain testing assays require approximately 30-min for all trials to be completed). In every experiment, the order of neuropeptide S doses tested was counterbalanced between rats to avoid potential order effects.

Thermal and Mechanical Pain Assays:

Hargreaves:

Applying the Hargreaves test/procedure took approximately 30 minutes in total(Avegno et al., 2018; Baiamonte et al., 2014; Hargreaves, 1988; Marendes et al., 2025). The assay applies radiant heat from a Hargreaves infrared (I.R.) power source (Ugo Basile, Italy, catalog number: 37570), by aiming a light beam to the middle of the plantar surface of the hindpaw. The intensity of the I.R. heat source was set to 60 I.R. units across all measures in this study (used to determine the rate of ramping-heat stimulus applied, adjustable in a range of 1–99 units in one-unit steps), previously determined to be the lowest setting at which animals reliably responded within the cut-off time(Marendes et al., 2025). When the rat retracts their paw, their paw withdrawal threshold (PWL) response was recorded beginning with the right hindpaw, left hindpaw, right hindpaw again, and left hindpaw again (4 trials total and 5 minutes between each trial). A cutoff time of 20 seconds was used to prevent paw tissue damage. In the case of non-contact responses (spontaneous locomotion of the rat), the rat was retested. Scores from each trial (4 trials) were averaged to yield the rats average PWL.

Robotic Von Frey:

Applying the robotic Von Frey test (also known as the dynamic plantar aesthesiometer) takes approximately 30 minutes in total(Marendes et al., 2025). The test is applied by aiming a probe from a robot device (Ugo Basile, Italy, catalog number: 37550), at the middle of the plantar surface of the hindpaw and increasing mechanical stimulation via computer-controlled ramping of the grams force (gf). When the rat retracts their paw, the gf was recorded beginning with the right hindpaw, left hindpaw, right hindpaw again, and left hindpaw again (4 trials total and 5 minutes between each trial). Scores from each trial (4 trials) were averaged to yield the rats average PWL. A cutoff of 50 gf (10-s ramp from 0–50 gf) was used as the cutoff to prevent paw tissue damage. In the case of non-contact responses (i.e., trial terminated prematurely by spontaneous locomotion of the rat), the rat was retested.

Electronic Von Frey:

Applying the handheld electronic von Frey test took approximately 30 minutes in total(Deuis et al., 2017; Marendes et al., 2025). The test is applied by aiming the probe attached to the handheld device (Ugo Basile, Italy, catalog number: 38450) at the middle of the plantar surface of the hindpaw and ramping the grams force (gf). When the rat retracts their paw, the gf was recorded beginning with the right hindpaw, left hindpaw, right hindpaw again, and left hindpaw again (4 trials total and 5 minutes between each trial). Scores from each trial (4 trials) were averaged to yield the rats average PWL. A cutoff time of 10 seconds was used to prevent paw tissue damage. In the case of non-contact responses (i.e., trial terminated prematurely by spontaneous locomotion of the rat), the rat was retested.

Manual Von Frey:

Applying the manual Von Frey test took approximately 30 minutes in total(Aguilar et al., 2024; Brandner et al., 2023; DTU, 2025; Marendes et al., 2025). The test is applied by aiming a Von Frey hair (Stoelting Touch Sensory Probes (Stoelting Co., Wood Dale, IL)) at a perpendicular angle to the plantar surface of hindpaw with sufficient force to slightly bend the filament and count 3 seconds. Testing occurred with eight fibers (rat; 8.0 g, 10 g, 15 g, 26 g, 60 g, 100 g, 180 g, 300 g), with the #4 filament (26 grams) always occurring first. If the paw withdraws after the stimulation (“stamping” of the paw or licking) then a positive response was recorded (“X”). If no response a negative response (“O”) was recorded. A positive response was followed with the next smallest filament and a negative response with the next largest filament. Following the first change in response sequence (i.e. O to X or vice versa), four additional measures were recorded, descending in fiber grams force after a response or ascending in fiber grams force after no response. The final response/no-response (“X/O”) sequence (e.g. “XXOXOXO” or “OOOXOXOO”) was used to estimate the 50% paw withdrawal threshold (in gf), calculated using a freely available calculator provided by Technical University of Denmark(DTU, 2025). In the case of non-contact responses (i.e., trial terminated prematurely by spontaneous locomotion of the rat), the rat was retested.

Experimental Design Overview:

Apparatus Habituation and Baseline Testing:

Male and female cohorts of rats were tested using the same design (see Fig. 1 for overview). First, all rats received ICV cannulation surgery and given 3 weeks recover before they acclimated to the testing environment, including thorough habituation to the Hargreaves testing arena and glass-floor platform. A limitation of our previous study(Marendes et al., 2025) is that we did not habituate the rats specifically to the grid floor used in conducting the Von Frey assay or monitor mechanical nociception threshold over time using the Von Frey assay, which could help explain why we did not reliably detect mechanical pain threshold sensitization (compared to thermal pain threshold sensitization) in our CIEV rats. Thus, in this experiment, we habituated the animals to the glass and grid floors for three days of 60-minute sessions before the baseline measures were taken, to ensure familiarity with both apparatus. Rats were then baselined for two sessions on the thermal Hargreaves to determine their average baseline paw withdrawal latency (PWL), and split into two groups (i.e., AD and alcohol-naïve controls) that were matched in terms of their baseline thermal nociception threshold.

Fig. 1). Experimental Timeline.

Overview of experimental design. Experimental groups included male and female rats. After rats were split into two groups, for remaining experimental stages, rats in the CIEV group were housed in alcohol-vapor chambers and exposed to daily cycles of alcohol vapor (14h:10h, ON:OFF), while the alcohol-naïve group remained exposed only to air (24h; i.e., CIEV vs. air exposure conditions maintained through experimental stages with background shaded orange).

Chronic Intermittent Ethanol Vapor Exposure (CIEV) and Blood Alcohol Levels (BALs):

Beginning at 12 weeks of age, alcohol-naïve controls were exposed to air without alcohol and AD rats were exposed to chronic intermittent ethanol vapor (CIEV) exposure in daily cycles designed to cause intoxication (14h:10h, ON:OFF; target 150mg/dL blood alcohol levels), for five weeks of pain threshold tracking and the duration of experiments (Exp. 1–3). At no point in the study were the rats in either group provided access to oral alcohol.

CIEV exposure was administered via alcohol vapor machines previously described (see(Marendes et al., 2025) for pictures of the custom machines). The critical machine components have been detailed at length in excellent methods papers (see(Avegno & Gilpin, 2019; Gilpin et al., 2008; O’Dell et al., 2004)) and match the machine and methods we have previously employed to study oxytocin’s effects in alcohol dependent rats (see(Marendes et al., 2025; Tunstall et al., 2019)). Nevertheless, in brief; The CIEV exposure machines consist of 6 compartments for housing rats (each measuring 76cm length × 68.5 cm width × 35.5cm; all walls 0.5cm thick). Rats were housed within standard rat housing cages (these cages measure 47.5 cm long × 25.5 cm wide × 21.5 cm high, including the wire-top lid) that were placed inside each CIEV exposure compartment (3 rodent cages fit into each alcohol vapor exposure compartment, and so 18 total rodent cages fit into each 6 compartment CIEV exposure machine). Males and females were housed in separate machines, and the ethanol vapor concentration was increased gradually over days/cycles of exposure to induce alcohol tolerance and dependence by increasing the “stroke rate” on the pump that supplied 95% ethanol (Decon Labs, Prussia, PA) to the warmed Erlenmeyer flask used to generate alcohol vapor (see(Marendes et al., 2025)) to achieve target blood alcohol levels (BALs) (150 mg/dL) for all rats (note: male and female rats are exposed to different stroke rates/concentrations of alcohol vapor [male>female] to achieve the same target BAL after compensating for their different physiology (primarily, their different bodyweights). BALs were assessed by collecting tail-blood samples at the end of 14-h ethanol vapor exposure periods and measuring alcohol concentration using an Analox AM1 analyzer (Analox Instruments Ltd, Stokesley, North Yorkshire, England). We ensure that we test a subset of rats that represent the full range of body weights in the cohort (across studies, we have noted that body weight correlates with resulting BALs) and that represent each compartment of the ethanol-vapor machine (across studies, we have noted that the compartment the rat is housed in does not correlated with resulting BALs in our ethanol-vapor machine, i.e., the concentration of ethanol vapor across compartments is consistent).

Tracking Thermal and Mechanical Nociception in Alcohol Dependence:

Beginning at 13 weeks of age, rats were tested in both the Hargreaves and robotic Von Frey assay once a week (on separate days of the week, with counterbalanced assay order), 6–8 hours into alcohol withdrawal to track the emergence of AD-induce hyperalgesia on the thermal and mechanical pain measures (i.e., increased pain sensitivity in the AD rats). Once the AD group developed a significantly quicker PWL response in both assays, we progressed to the NPS testing phase.

Stylet-Clean Tests:

To habituate rats to the handling and ICV-injection procedure in a gradual manner, on separate days before proceeding to any pharmacological tests, we first measured the impact of stylet-cleaning alone on each nociception assays (i.e., “stylet-clean test”). The stylet-clean manipulation involved removing the 6mm custom stylet from the implanted ICV cannula using curved-tip tweezers, cleaning the stylet with a 70% ethanol pad, and returning the –air dried– stylet to position (i.e., the same procedure to be used during later pharmacological testing, but without any ICV administration). The rats were placed into the testing apparatus and left undisturbed for 5 minutes to simulate the pretreatment time that would be used in subsequent pharmacological tests, before conducting a pain measurement (this handling and injection habituation was performed in the same manner prior to pharmacological testing using the Hargreaves and robotic Von Frey assays). All rats underwent handling and stylet-clean habituation for two tests and once it was determined that handling and stylet cleaning before pain testing did not significantly alter their PWL, NPS testing began.

NPS Testing in Alcohol Dependence:

Beginning at 17 weeks of age, rats underwent a series of pharmacological tests. We administered ICV injections of NPS or saline 5 minutes before the Hargreaves or robotic Von Frey test. A 5-minute pretreatment time was selected to ensure that nociception measures coincided with the time course of NPS’s effects (see Supplement File 1 for a time course of NPS’s enhancement of locomotion, assessed in a separate cohort of rats).

Comparison of Mechanical Assays:

After testing the effects on NPS on thermal and mechanical hyperalgesia, a follow-up experiment was conducted to directly compare three methods for applying the Von Frey test. In our earlier work, the DPA assay failed to reliably detect AD-induced hyperalgesia, potentially due to insufficient habituation or stress associated with the testing apparatus. However, in this study we used a thorough procedure to habituate the animals to the testing environment prior to testing. Given the success in detecting a stable AD-induced mechanical hyperalgesia in both male and female cohorts in this experiment, we decided to determine the robustness of this effect and generalizability across mechanical nociception assay type, by directly comparing mechanical sensitivity across tests and thereby resolve an unanswered question from our previous work. Using the same mechanical tests as in our published work(Marendes et al., 2025), we compared the electronic, robotic, and manual Von Frey assays. Each test was counterbalanced across 3 sessions (never on consecutive days) to minimize potential order effects.

Data and Statistics:

Data were analyzed and plotted in Prism (GraphPad Prism 10). Statistical analyses were completed using t-tests (paired or unpaired as appropriate) and 3-Way or 2-Way ANOVAs (Repeated or Mixed Measures as appropriate), with significant main effects or interactions followed by Holms-Sidak post-hoc tests. Furthermore, treatment doses were compared to the control condition using a many-to-one approach for post-hoc testing (i.e., 0.1 nmol and 1 nmol NPS doses compared to the saline control). Statistical significance was set at p < 0.05. All raw data are provided in a Supplemental File 5 accompanying the manuscript. All main manuscript figures and statistics represent data analyzed after collapsing across sex. All data was also analyzed including Sex as a factor, and plotted separately for male and female rats (see Supplemental File 2–4).

Results:

Apparatus Habituation and Baseline Testing:

While habituating rats to the glass and grid floors before collecting their thermal baseline, we monitored their behavior and noticed a behavior that we had previously neglected; rats initially took a splayed posture (approximately 1 min) and then continued to not walk/explore normally for an additional approximately 10 min. This was observed to occur at the start of each habituation period, would appear again in switching between glass and grid floor (particularly when exposed to the grid floor), and this behavior would decrease over time and with repeated exposures. Once all rats no longer displayed this behavior in response to being placed in the arena on either grid floor (i.e., behavior was extinguished over approximately 3 exposures), only then did we move on to measuring their thermal baseline nociception threshold using the Hargreaves assay.

Tracking Thermal and Mechanical Nociception in Alcohol Dependence:

The male and female rats assigned to the AD (n=9) and alcohol-naïve controls (n=9) were well matched in terms of their pre-CIEV exposure Hargreaves PWL response (Fig. 2A; t (17) = 0.02489, p = 0.9804) (see data split by Sex in Supplemental File 2). The AD and alcohol-naïve control rats were tracked weekly on the Hargreaves and robotic Von Frey to determine the emergence of AD-induced hyperalgesia in AD animals for both thermal and mechanical pain. The average male (n= 3) BALs were 172.6 (± 40.67) mg/dL and the average female (n= 4) BALs were 184.4 (± 23.15) mg/dL; the terminal stroke rate that was maintained for the duration of experiments after hitting this level. After three weeks of testing, the AD developed AD-induced hyperalgesia with a significant main effect of Vapor Exposure for Hargreaves (Fig. 2B; F (1, 17) = 21.01, p = 0.0003), and robotic Von Frey (Fig. 2C; F (1, 17) = 12.40, p = 0.0026), with no main effect of Session (Fig. 2B; F (2, 34) = 0.1903, p = 0.8276) (Fig. 2C; F (2, 34) = 1.122, p = 0.3373) and no interaction (Fig. 2B; F (2, 34) = 0.7183; p = 0.4948) 2C; F (2, 34) = 0.3239; p = 0.7255). The quicker PWL response time in the AD in both the on Hargreaves and robotic Von Frey assays compared to alcohol-naïve controls, indicated a stable sensitization of pain in the AD rats across thermal and mechanical modalities.

Fig. 2). Pain Baseline and Tracking Thermal and Mechanical Nociception Threshold.

Rats were initially matched in terms of baseline paw withdrawal latency (PWL) in the Hargreaves thermal nociception assay (A). Tracking rats thermal and mechanical nociception threshold with once a week Hargreave’s (B) and Von Frey (C) assays indicated that alcohol exposure induced hyperalgesia in both modalities assessed. (see data split by Sex in Supplemental File 2).

Stylet-Clean Tests:

Before NPS testing on the thermal (Fig. 3A) and mechanical (Fig. 3B) pain measures, the rats received two stylet-clean tests (see methods) to determine if handling stress including removal of the stylet altered behavioral responses. The stylet-clean tests were not significantly different from the prior week 3 Hargreaves and robotic Von Frey measurements (Fig. 2B & 2C) with no main effect of Session for Hargreaves (F (2, 34) = 0.05055, p = 0.9508) and robotic Von Frey (F (1.161, 18.58) = 0.09157, p = 0.8026) assays, and a main effect of Vapor Exposure for both Hargreaves (F (1, 17) = 27.93, p < 0.0001) and robotic Von Frey (F (1, 16) = 8.571, p = 0.0099).

Fig. 3). Testing ICV NPS on Hargreaves and Von Frey.

Neuropeptide S (NPS) produced analgesia in the thermal Hargreaves (A) and mechanical Von Frey (B) assays. This effect was observed independent of alcohol dependence, and was similar in male and female rats (Supplemental File 3). Nevertheless, NPS produces sufficient analgesia to alleviate pain sensitivity in hyperalgesic AD rats to the to/beyond the level of sensitivity observed in vehicle treated, alcohol-naïve controls (dashed line). (see data split by Sex in Supplemental File 3).

NPS Testing in Alcohol Dependence:

In testing ICV treatments on the Hargreaves measure (Fig. 3A), we observe again the significant main effect of Vapor Exposure (Fig. 3A; F (1, 17) = 13.69, p= 0.0018), a main effect of NPS Treatment (Fig. 3A; F (2, 34) = 40.27, p= 0.0001), but no significant interaction of Vapor Exposure X NPS Treatment (Fig. 3A; F (2, 34) = 0.5144, p = 0.6025) (see data split by Sex in Supplemental File 3). Both 0.1 nmol and 1 nmol NPS doses (Fig. 3A; ps < 0.0001) increased PWL in AD and alcohol-naïve controls, compared to saline.

After the testing of Hargreaves with NPS administration (Fig. 3A) and before the testing of robotic Von Frey with NPS administration (Fig. 3B), one female dropped out of the dependent group and was excluded from the experiment due to health complications of unknown origin. Their data does not contribute to statistical analyses in Figures 3B and 4A–C and is not presented in the Supplemental File 5 raw data file. In testing ICV treatments on the robotic Von Frey measure (Fig. 3B), we observe again the significant main effect of Vapor Exposure (Fig. 3B; F (1, 16) = 8.477, p= 0.0102), a main effect of NPS Treatment (Fig. 3B; F (2, 32) = 26.09, p < 0.0001), but no significant interaction of Vapor Exposure X NPS Treatment (Fig. 3B; F (2, 32) = 1.888, p = 0.1679). NPS at both 0.1 nmol (Fig. 3B; p = 0.0004) and 1 nmol doses (Fig. 3B; p < 0.0001) increased PWL in AD and alcohol-naïve controls, compared to saline.

Figure 4). Comparison of Alcohol-Dependence-Induced Hyperalgesia Across Three Von Frey Mechanical Pain Assays.

Alcohol-dependence-induced hyperalgesia that was first detected in the robotic Von Fry assay (also known as the dynamic plantar aesthesiometer; Fig. 3B) generalized across counterbalanced independent tests using the handheld-electronic (A), robotic (B), and manual (C) Von Frey assays of mechanical nociception threshold. In all three assays, pain sensitivity in hyperalgesic AD rats was significantly more sensitive than the threshold observed in alcohol-naïve controls (dashed line). (see data split by Sex in Supplemental File 4).

Comparison of Mechanical Assays:

Following up on an experiment in our previously published work(Marendes et al., 2025), we wanted to determine whether detecting mechanical hyperalgesia in AD compared to alcohol-naïve controls was contingent on the method used to assess mechanical threshold. This time using both male and female rats, for the last experiment we compared the electronic, robotic, and manual von Frey tests, counterbalanced to prevent potential order effects. When we compared AD and alcohol-naïve controls using the electronic Von Frey, we found a significant difference between the groups (Fig. 4A; t (18) = 2.931, p = 0.0099) (see data split by Sex in Supplemental File 4). When we compared the AD and alcohol-naïve controls using the robotic Von Frey, we observed a significant difference between the groups grams force (gf) (Fig. 4B; t (18) = 2.516, p = 0.0262). Lastly, we also found a significant difference when we compared the groups on the manual Von Frey (Fig. 4C; t (18) = 3.265, p = 0.0056).

Discussion:

Together, these data demonstrate that AD produces increased pain sensitivity (i.e., hyperalgesia) in both male and female rats in both thermal and mechanical assays. Further, central administration of NPS produced analgesia across sex and across pain assays, independent of alcohol-exposure history (but sufficient to overcome the hyperalgesia that developed in AD rats). After monitoring rats behavior to encourage sufficient habituation to both the glass and grid floors, we were able to reliably detect both thermal and mechanical pain sensitivity, thereby resolving an uncertainty from our prior work(Marendes et al., 2025). Importantly, we further confirmed the robustness of AD-induced mechanical hyperalgesia by demonstrating this observation generalized across three independent, commonly-used assays to assess mechanical nociception threshold.

We hypothesized that NPS could exert an analgesic effect that could ameliorate the hyperalgesia that developed in AD rats. NPS was effective at promoting analgesia in both AD and alcohol-naïve controls, suggesting that the effect was not specific to the pathological state of alcohol dependence. This partly challenged our hypothesis, as we anticipated that CIEV-exposure would alter NPS receptor (NPSR1) function in a way that would render AD animals uniquely sensitive to exogenous NPS(Cannella et al., 2022; Ruggeri et al., 2010). It is important to consider at this stage the possibility that non-specific effects of NPS may have contributed to the analgesia measured, such as sedation or motor impairment. While this may be a concern in testing most analgesic compounds, that typically have sedative effects at higher doses (e.g., mu-opioid acting compounds such as morphine(Erichsen et al., 2005; Hestehave et al., 2019) and other anti-stress peptides such as oxytocin(Marendes et al., 2025; Peters et al., 2017)), NPS is quite unique in that it actually has analgesic effects at doses that simultaneously enhances locomotion/arousal (Supplement File 1; Okamura & Reinscheid, 2007; Reinscheid et al., 2005; Xu et al., 2004). In testing the effect of central NPS on paw withdrawal latency (PWL; i.e., in either thermal Hargreave’s or mechanical Von Frey assays), we would expect then the locomotion/arousal enhancing effects of NPS to accelerate the PWL response to the pain stimulus, resulting in false-positive pain responses and detection of a lower pain threshold following NPS administration. However, what we actually observed was a slower response to pain stimuli (i.e., a higher pain threshold, indicating analgesia) upon central NPS administration, making it very unlikely that non-specific effects of NPS could explain the analgesic effects observed. Together, this suggests that although NPS’s analgesic effects were not specific to the AD group as originally hypothesized, the analgesic effect of NPS was robust and repeatable across alcohol history, sex, and nociception assay employed. This generalizability may actually add more than detract from support for the idea of NPS as a therapeutic in pain states and related conditions (e.g. anxiety and alcohol/substance abuse).

An important outcome of this work was finding a solution to the challenge that had previously made it difficult to detect AD-induced hyperalgesia using Von Frey(Marendes et al., 2025). Although there was precedent for this observation, we were surprised to discover the degree to which the type of floor the rats were habituated to appears able to influence the nociceptive responses(Deuis et al., 2017; Mogil, 2009). When the rats were first placed on a novel grid or glass floor, they exhibited a splayed-feet, cautious posture, suggesting an stress/avoidance response. This behavior likely masked pain sensitivity by elevating baseline paw withdrawal thresholds and in general increasing variability in responses. However, through repeated habituation sessions we observed that rats gradually adapted to the testing environment, eventually entering the chambers and moving freely without hesitation. Once this habituation was achieved, we were able to consistently detect both thermal and mechanical hyperalgesia across assays. In reviewing the literature, it was difficult to find details of this aspect of the habituation procedures, although it appears important to the sensitivity of assays employed and could help explain differences in results reported by different groups. Therefore, future studies would benefit from monitoring the habituating of animals to the test arena/s prior to pain testing, to minimize neophobia-related variability.

The present experimental design also provides us the opportunity to directly compare the behavior of male and female rats in all assays. We were interested in understanding potential sex differences in rats’ sensitivity to pain, development of CIEV-induced hyperalgesia, the effect from NPS administration, and generalization of CIEV-induced hyperalgesia across different mechanical pain modalities. As shown in Supplement File 2–4 (all data appears analyzed with Sex included as a factor) male and female rats responded similarly in most measures. Supplement File 2 shows baseline and longitudinal pain sensitivity data separated by sex for both the Hargreaves and Von Frey assays. In the baseline measure, we observed a main effect of Sex (Fig. S2A) with males being more sensitive in the thermal nociception assay, concordant with the literature (Bourgeois et al., 2022), however, this sex difference was not persistent through longitudinal, repeated measurements (Figs. S2B and Fig. S2C). Further, no main effects or interactions involving Sex were observed in the development of CIEV-induced hyperalgesia, indicating that the development and level of hyperalgesia were equivalent between male and female rats (Figs. S2B and Fig. S2C). Similarly, Supplemental File 3 shows that the analgesic effects of NPS were not sex dependent. Finally, CIEV-induced hyperalgesia generalized between male and female rats across the three Von Frey mechanical assays that we compared (Supplemental File 4). Together, these findings demonstrate that AD-induced hyperalgesia and NPS’s analgesic effect were produced consistently across pain modality, similarly in male and female rats.

It is generally accepted that females are more sensitive than males in terms of behavioral response to mechanical nociceptive stimuli (Hendrich et al., 2012). However, it appears that at the same time, females counterintuitively have a higher threshold for activation of peripheral nociceptors by mechanical stimuli when compared to males (Hendrich et al., 2012). This suggests that central nervous system mechanisms are responsible for the greater sensitivity of behavioral response that has generally been reported in females. In the present study, we did not observe a sex difference in our measurement of mechanical nociception threshold, and this was consistent throughout the three mechanical nociception assays that we compared (see Supplementary File 2–4 for data split by Sex). It is difficult to compare this absence of sex-difference directly to previous work, as in this study we made a point of thoroughly habituating rats to the testing apparatus before assessing baseline mechanical nociception, and conducted longitudinal, repeated measurement of mechanical nociception (i.e., habituation to the apparatus and test procedure/stimulus likely reduced the contribution of central mechanisms that are believed to confer a sex difference in mechanical nociception, e.g., response to novel context/stimulus). In general, previous studies have typically employed single, acute assessments of nociception when noting sex differences, and future studies that monitor any observed sex difference over time could help to shed light on the stability and adaptability of such differences. It should be noted also, that our experimental design further complicates drawing direct comparison to this previous work, as rats were assessed for mechanical nociception threshold only after having experienced thermal nociception testing, surgery, and one week of exposure to chronic-intermittent ethanol vapor (i.e., these experiences may also have influenced baseline mechanical nociception and our ability to detect sex differences).

One potential limitation to consider in this study is that we baselined the rats on the thermal Hargreaves measure but not the mechanical Von Frey. The baseline Hargreaves measure was used to determine group assignment (i.e., rats were split into two evenly matched groups before being tracked on both pain assays). Therefore, we relied on random assignment to generate groups matched in terms of mechanical threshold; however, we do not have any reason to think groups of rats matched very well in thermal nociception threshold would not be well matched in mechanical nociception threshold.

Conclusion:

In summary, these data demonstrate that alcohol dependence produces hyperalgesia across both thermal and mechanical modalities in rats. Further, we demonstrate that NPS administration produced analgesia across both pain modalities, regardless of sex or alcohol-exposure history. While not in line with our original hypothesis, the observation that NPS was equally effective in alcohol dependent and alcohol-naïve control rats suggests that the analgesic action of NPS was not specific to the alcohol dependence state (i.e., analgesia rather than anti-hyperalgesia), these data nonetheless suggest NPS-NPSR1 interaction as a highly promising candidate for the treatment of pain conditions, including AD-induced pain, given the robustness and generalizability of analgesia produced by NPS administration, coupled with the lack of abuse potential associated with NPS-NPSR1 interaction. These findings lay a foundation for future studies aimed at dissecting the underlying NPS circuitry, which we hypothesize may also be relevant to curbing the motivation to seek and consume alcohol and other drugs (e.g., opioids).

Highlights:

• Male and female rats developed alcohol-dependence induced hyperalgesia in thermal (Hargreaves) and mechanical (Von Frey) assays.

• Central administration of Neuropeptide S (NPS) produced thermal and mechanical analgesia in both alcohol-dependent and alcohol-naïve rats.

• Male and female rats appeared similar in terms of both the development of hyperalgesia in alcohol dependence and the analgesic effect of NPS.

• The hyperalgesia observed in alcohol dependent rats generalized across three methods of applying the Von Frey test, and in comparing to our previous work, the importance of habituation in assaying nociception is discussed.