We first examined the effect of intravenous fentanyl to induce cardiorespiratory depression and activate neurons in the nucleus tractus solitarius (nTS). Catheterized rats received intravenous saline (1 ml/kg) or fentanyl at a range of doses (2, 20, or 50 µg/kg; Figure 1A), and cardiorespiratory parameters were measured up to 60 min. The first 20 min of these responses are shown in Figure 1B–D. As shown in Supplementary file 1A, baseline cardiorespiratory parameters were similar between male and female rats that received 20 µg/kg fentanyl. There was a small but significant difference in basal heart rate in female rats that received 50 µg/kg fentanyl (Supplementary file 1B). Neither saline nor 2 µg/kg fentanyl evoked a significant change in cardiorespiratory parameters. In comparison, both 20 and 50 µg/kg fentanyl produced rapid decreases in oxygen saturation, heart rate, and respiratory rate, with the largest decrease occurring after 50 µg/kg fentanyl. The nadir (defined as the lowest point measured after fentanyl) and recovery (calculated as 90% of baseline values prior to fentanyl) for each physiological parameter were assessed in all groups (Figure 1E–J). There was no significant difference in the nadir or recovery values between saline- and 2 µg/kg fentanyl-treated rats. In comparison, rats given 50 µg/kg fentanyl exhibited a significantly lower nadir in oxygen saturation (Figure 1E) and respiratory rate (Figure 1I) compared to 20 µg/kg. In addition, the recovery time was significantly longer in rats that received 50 µg/kg fentanyl compared to 20 µg/kg fentanyl (Figure 1F, J). The nadir of bradycardia was similar between 20 and 50 µg/kg rats (Figure 1G), although the duration of the response was significantly longer in rats that received the highest dose of fentanyl (Figure 1H). Overall, we observed no sex differences in the nadir or recovery time in rats that received 20 µg/kg fentanyl. Female rats that received 50 µg/kg fentanyl exhibited a significantly longer recovery of respiratory rate, although oxygen saturation and heart rate recover times were similar to males (Supplementary file 1C, D). We next examined nTS activation (Fos-IR) following intravenous fentanyl (Figure 1K). A significant increase in Fos-IR was observed after 2 µg/kg fentanyl (Figure 1L), a dose that did not induce cardiorespiratory depression. Further dose-dependent increases in Fos-IR were observed after 20 and 50 µg/kg fentanyl. We evaluated a subpopulation of catecholaminergic nTS neurons, which are critical for full expression of hypoxia-evoked cardiorespiratory responses (King et al., 2012; King et al., 2015) for activation after intravenous fentanyl (Figure 1M). These cells exhibited increased Fos-IR after 2 µg/kg fentanyl and were further activated at the highest dose of fentanyl (50 µg/kg).

The above data provide a comprehensive assessment of the dose-dependent changes in cardiorespiratory parameters and nTS neuronal activation evoked by intravenous fentanyl. To gain insight into the extent to which central and peripheral opioid receptors mediate these effects, separate cohorts of rats received intravenous administration of the centrally and peripherally acting competitive opioid receptor antagonist naloxone (NLX; 1 mg/kg) or the peripherally restricted opioid receptor antagonist naloxone methiodide (NLXM, 1 and 5 mg/kg). Initial biodistribution experiments were performed to confirm the peripheral restriction of NLXM. Catheterized rats that received intravenous NLXM (1 or 5 mg/kg) or NLX (1 mg/kg) were evaluated for detection of NLX or NLXM in brain and plasma samples collected either 2 or 10 min after administration (Figure 2A). Neither dose of NLXM was detected within the central nervous system at either time point evaluated (Figure 2B, C). Importantly, NLX was not detected in these NLXM-treated groups. In comparison, rats that received intravenous NLX exhibited significant, time-dependent detection of NLX in both plasma and brain samples (Figure 2D). Thus our data are in agreement with previous reports confirming the peripheral restriction of NLXM (Perekopskiy et al., 2020; McGaraughty et al., 2005). We next evaluated cardiorespiratory changes from baseline in rats that received intravenous NLX or NLXM at either dose (Figure 2—figure supplement 1A–D). Data indicate that neither NLX nor NLXM significantly altered cardiorespiratory parameters from their baseline parameters (Figure 2—figure supplement 1E–J). Based on these findings, we utilized NLXM to further investigate the roles of peripheral MOR antagonism in the context of evaluating OIRD.

Catheterized rats received intravenous NLXM (1 or 5 mg/kg) or NLX (1 mg/kg) prior to intravenous fentanyl administration at 20 µg/kg (Figure 3A) or 50 µg/kg (Figure 3—figure supplement 1A). As shown in Figure 3B–D and Figure 3—figure supplement 1B–D, the responses evoked by 20 and 50 µg/kg fentanyl induced an onset and duration of cardiorespiratory depression that was similar to what was observed in Figure 1. As expected, NLX pretreatment completely blocked the decrease in cardiorespiratory parameters at both 20 and 50 µg/kg doses of fentanyl. In rats that received 20 µg/kg fentanyl, 1 mg/kg NLXM pretreatment prevented bradycardic and hypoventilatory responses to fentanyl (Figure 3B–D). For all parameters, saline-pretreated rats exhibited lower nadir values than NLX- and NLXM-pretreated rats, and there was no significant difference in the nadir or duration between NLX (1 mg/kg) and NLXM (1 mg/kg) pretreated rats that received 20 µg/kg fentanyl (Figure 3E–J). Although there was a small drop in the nadir of oxygen saturation in NLXM-pretreated rats, this was not significantly different than NLX-pretreated animals (Figure 3E, p = 0.2292). In rats that received 50 µg/kg fentanyl, 1 mg/kg NLXM pretreatment was unable to prevent the initial decrease in oxygen saturation and heart rate (Figure 3—figure supplement 1E, G). However, the duration of these responses was significantly shorter compared to saline-treated rats (Figure 3—figure supplement 1F, H, J). A higher dose of NLXM (5 mg/kg), which does not cross the blood–brain barrier (Figure 2C), was then used in a separate group of animals that received 50 µg/kg fentanyl. Pretreatment with 5 mg/kg NLXM attenuated cardiorespiratory depression. While the nadir of oxygen saturation was significantly lower than NLX-pretreated rats, the duration of this effect was not significantly different between groups (Figure 3—figure supplement 1E–J). No sex differences were observed in the nadir or recovery time of any parameter (Supplementary file 1C, D). Together, these data demonstrate that peripheral MOR antagonism can sufficiently prevent OIRD by 20 µg/kg fentanyl and strongly attenuates OIRD evoked by 50 µg/kg fentanyl.

Neuronal activation, as measured by Fos-IR, was examined in the nTS of rats pretreated with saline or opioid receptor antagonists followed by 20 µg/kg (Figure 3K) or 50 µg/kg fentanyl (Figure 3—figure supplement 1K). Saline pretreatment evoked the highest degree of fentanyl-induced Fos-IR in the nTS, including in a subpopulation of TH-IR neurons. Blocking peripheral MOR signaling with NLXM did not decrease fentanyl-induced nTS neuronal activation, while NLX, which blocks both central and peripheral MORs, resulted in a significant reduction in nTS activation (Figure 3L, M). In rats that received the highest dose of fentanyl, the number of nTS cells displaying Fos-IR was similar between rats pretreated with saline and NLXM at both doses, with the only significant reduction occurring in NLX-pretreated animals (Figure 3—figure supplement 1L). This degree of activation was also observed in catecholaminergic cells (Figure 3—figure supplement 1M), although around 30% of TH-IR neurons also displayed Fos-IR in NLX-pretreated rats.

The above data demonstrate that peripheral MOR antagonism prevents fentanyl-induced respiratory depression without affecting nTS neuronal activation as measured by Fos-IR. Given that reversal of OIRD has more translational relevance, we next evaluated the extent to which NLXM reverses cardiorespiratory depression after it has been induced by 20 µg/kg (Figure 4A) or 50 µg/kg fentanyl (Figure 4—figure supplement 1A). Both 20 (Figure 4B–D) and 50 µg/kg fentanyl (Figure 4—figure supplement 1B–D) induced rapid decreases in oxygen saturation, heart rate, and respiratory rate, similar to what is observed in Figure 1. The fentanyl-induced nadir in all physiological parameters prior to intravenous administration of saline, NLX, or NLXM was similar between groups. In rats that received 20 µg/kg fentanyl, both intravenous NLX (1 mg/kg) and NLXM (1 mg/kg) restored cardiorespiratory values to baseline significantly faster than saline-pretreated animals (Figure 4F, H, J). This reversal of cardiorespiratory parameters was similar between NLX- and NLXM-treated animals. In rats that received 50 µg/kg fentanyl, NLX rapidly restored all physiological parameters to their baseline values (Figure 4—figure supplement 1F, H, J). While 1 mg/kg NLXM did not sufficiently reverse the OIRD induced by 50 µg/kg fentanyl (Figure 4—figure supplement 1F), a higher dose of 5 mg/kg NLXM fully reversed the fentanyl-induced bradycardia and hypoventilation. The time to restore parameters to baseline was not significantly different than NLX. We did not observe any sex differences in the nadir or recovery time of any parameter (Supplementary file 1C, D). Thus, blocking peripheral MORs sufficiently reverses OIRD at both moderate and high doses of fentanyl.

Because acute hypoxia activates nTS neurons (Teppema et al., 1997), we investigated the impact of peripheral and central MOR antagonism on fentanyl-induced nTS neuronal activation (Figure 4K). At both doses of fentanyl administered, a similar degree of nTS neuronal activation was observed in all groups regardless of MOR antagonist administered after fentanyl (Figure 4L, M; Figure 4—figure supplement 1L, M). Together, these data demonstrate that selective antagonism of peripheral opioid receptors sufficiently reverses cardiorespiratory depression induced by both moderate and high doses of fentanyl. Moreover, these data suggest that even a brief period of fentanyl-induced cardiorespiratory depression is sufficient to induce robust activation of nTS neurons, and this subsequent MOR antagonism has no additional effect to increase nTS activation.

NLX rapidly reverses OIRD, but produces unwanted side effects including the immediate onset of severe withdrawal (Lewanowitsch and Irvine, 2002; Lai et al., 2021). The intense symptoms induced during NLX-precipitated withdrawal present additional challenges in managing OUD in patients. Given that peripheral MOR antagonism sufficiently reverses fentanyl-induced cardiorespiratory depression to a degree that is comparable to NLX, we used a conditioned place aversion (CPA) approach to assess whether selective peripheral antagonism of MOR signaling produces aversive behaviors. Rats underwent a three-session CPA challenge that consisted of pre-conditioning test, conditioning test, and post-conditioning test (Figure 5A). Horizontal locomotion in both compartments of the CPA boxes was assessed during both pre- and post-conditioning tests (Figure 5B). During the pre-conditioning test, rats exhibited no natural preference or aversion for either compartment. On the conditioning day (Day 2), a divider was placed in the middle of the CPA box. Rats received intravenous fentanyl (50 µg/kg) in their home cage. Four minutes later, rats were placed into the paired compartment of the CPA box and immediately given intravenous saline (1 ml/kg), NLX (1 mg/kg), or NLXM (1 or 5 mg/kg). During the post-conditioning test, the divider was removed, and rats were allowed to explore both compartments of the CPA box. Figure 5C shows the time spent on the conditioned side of the CPA box for all groups. During the post-conditioning test, NLX-treated animals spent significantly less time in the NLX-paired compartment, suggesting that the reversal of OIRD on the conditioning day was aversive. This CPA behavior was not observed in rats that received NLXM at either dose (Figure 5D). Importantly, a single conditioning session with fentanyl administered in the home cage followed by saline in the compartment instead of an MOR antagonist did not produce a preference or aversion to the saline-paired compartment. To rule out any potential effects of endogenous opioids influencing the behavioral responses to opioid receptor antagonism, we performed an additional test in which rats were given saline in their home cage, followed by NLX or NLXM in one of the CPA compartments (Figure 5E, F). Neither antagonist produced a preference nor aversion to the MOR antagonist-paired compartment (Figure 5G). There was no difference in CPA scores between these groups (Figure 5H). These findings are consistent with our data demonstrating that NLX and NLXM alone do not alter physiological parameters from baseline (Figure 2—figure supplement 1).

As shown in Figure 1, fentanyl-induced robust Fos-IR expression in the nTS. While Fos-IR quantification provides insight into the anatomical activation of brainstem subregions in response to fentanyl administration and OIRD reversal, this approach does not provide any temporal resolution of the activity of these cells. Therefore, we used wireless in vivo fiber photometry to assess dynamic changes in nTS calcium transient activity after intravenous fentanyl administration in anesthetized rats (Figure 6A). Catheterized rats underwent bilateral injections of an AAV expressing a calcium sensor (pGP-AAV9-syn-jGCaMP8m-WPRE) into the nTS. On the day of the experiment, an optic fiber was lowered on the surface of the brainstem above the nTS and calcium transient activity was recorded (Figure 6B). GCaMP viral expression was observed throughout the caudal-rostral extent of the nTS in all rats (Figure 6C). Fentanyl (20 µg/kg) infusion induced a biphasic activity profile in nTS neurons, characterized by an initial robust increase (Early Response) followed by a decrease below baseline (Late Response, Figure 6D). Pretreatment with NLXM (5 mg/kg) attenuated both phases of the fentanyl-induced changes in neuronal activity. As shown in Figure 6E, the area under the curve (AUC) was significantly elevated during the Early and Late Responses after fentanyl. In contrast, no significant fentanyl-induced change in AUC was observed in NLXM-pretreated rats at either time point, indicating that changes in fentanyl-induced nTS neuronal activity are strongly mediated by peripheral MORs. We next examined the extent to which MOR antagonists restore the fentanyl-induced reductions in activity. After MOR antagonist administration, fentanyl-induced suppression of nTS activity returned to baseline during the early recovery in rats that received NLX, but not NLXM. However, both NLX and NLXM enhanced nTS activity above baseline during the late recovery period (Figure 6F, G). During the Late Recovery period, nTS activity was significantly enhanced above baseline in both NLX- and NLXM-treated animals. Together, these data demonstrate that peripheral MOR antagonism is sufficient to reverse the fentanyl-induced decrease in nTS neuronal activity.

Animals were deeply anesthetized using isoflurane (3% induction, 2% maintenance). A small incision was made in the dorsal surface of the neck. Another small incision was made on the ventral surface of the neck, and underlying tissue was carefully dissected to expose the jugular vein. An indwelling catheter was inserted into the jugular vein and sutured in place using non-absorbable silk suture. The catheter was then tunneled subcutaneously and exited the body through the first small hole in neck on the dorsal side. The ventral incision was closed using non-absorbable silk suture. The exposed catheter was connected to a backpack device (Instech) containing a port for drug administration. Animals were given Carprofen (2 mg/kg sc), Baytril (8 mg/kg sc), and bupivacaine (5 mg/kg, site of incision). In addition, rats were given Carprofen tablets (Bio Serv, MD150-2) for 2 days after surgery to assist in wound healing and analgesia. Rats were singly housed following surgery and were allowed to recover for 1 week prior to random assignment to an experimental group. Catheter patency was maintained with daily flushing of 0.3 ml sterile saline containing gentamicin (1.33 mg/ml, i.v.). Rats with a loss of catheter patency were excluded from the study.

Following recovery from IV catheterization surgery, conscious, freely moving rats in their home cages were fitted with a non-invasive pulse oximeter collar (Starr LifeSciences). Cardiorespiratory parameters (oxygen saturation, heart rate and respiratory rate) were collected (MouseOx v2.0, Starr LifeSciences) at baseline conditions in animals breathing room air, and after intravenous administration of fentanyl citrate (2, 20, or 50 µg/kg). A subset of animals received weight-adjusted intravenous administration of the nonselective opioid receptor antagonist naloxone hydrochloride (NLX; 1 mg/kg; Sigma, Cat#: N7758, CAS: 51481-60-8) or the peripherally restricted opioid receptor antagonist naloxone methiodide (NLXM; 1 or 5 mg/kg; Sigma, Cat#: N129, CAS: 93302-47-7) before or after fentanyl administration. Cardiorespiratory parameters were then measured for up to 60 min. For all OIRD experiments, the time course graphs consist of 30 s of data averaged into a single data point. The nadir was defined as the lowest point measured after fentanyl administration (for all graphs, red arrow indicates time of fentanyl administration, time = 0 min). In addition, the recovery rate was defined as the time to reach 90% baseline of pre-fentanyl values for each cardiorespiratory parameter. Two hours after fentanyl administration, rats were transcardially perfused with 0.01 M PBS followed by paraformaldehyde (4% in 0.01 M PBS). Brains were removed and stored overnight at 4°C in 4% paraformaldehyde for post-fixation, followed by at least 72-hr incubation in sucrose (30% in 0.01 M PBS) solution. Isopentane was used to flash-freeze brains. Brainstem sections containing the nTS were sliced in the coronal plane (40 µm) using a cryostat (Leica CM 1950).

A subset of previously catheterized drug-naive rats was deeply anesthetized and then received intravenous administration of NLX (1 mg/kg) or NLXM (1 or 5 mg/kg). While still under anesthesia, rats were quickly decapitated 2 or 10 min after intravenous administration of MOR antagonists. MOR antagonists were dosed in all rats at an experimentally relevant concentration with triplicate blood and brain samples collected at two time points. The early time point at 2 min evaluated conditions when the plasma concentration of the OR antagonist is high and reflects the identical time point of intravenous administration for OIRD pretreatment experiments. The second collection time point (10 min) was intended to evaluate steady-state conditions and represent physiological experiments when reversal of OIRD was achieved by both antagonists. Brains were quickly removed and flash frozen in liquid nitrogen. Trunk blood was collected into capillary collection tubes containing lithium heparin (Greiner Bio-One; Fisher Scientific). The blood was spun on a centrifuge for 10 min at 4°C, and plasma was carefully extracted from the supernatant. Brains and plasma samples underwent PK analyses for detection of NLX or NLXM at both time points. Standard rat PK studies dosed the candidate MOR antagonists and sample blood over multiple time points. A general description of tissue distribution can be obtained during PK modeling by calculating the volume of distribution. Samples underwent a direct measurement of peripheral restriction by measuring compound concentration in plasma and brain.

The aversive effects following reversal of OIRD were measured using CPA (Tzschentke, 2007) using the video tracking system ANY-maze (RRID:SCR_014289). During a pre-conditioning session (Day 1), rats were allowed to explore both sides of a two compartment CPA box for 15 min. On the Conditioning Day (Day 2), a divider was placed in the middle of the box to restrict movement to one side on the box. Rats were then randomly assigned to one side of the CPA box, and this side was paired with either saline (1 ml/kg), NLX (1 mg/kg), or NLXM (1 or 5 mg/kg). Rats were given intravenous fentanyl (50 µg/kg) or saline in their home cages. Four minutes later, they were placed in the paired side of the box and immediately given saline or an MOR antagonist. Rats were confined to this compartment for 15 min. For all CPA experiments, measures were taken to balance the side of CPA box by drug and by sex. On the post-conditioning test (Day 3), the divider was removed. Rats were then placed in the CPA box and allowed to explore both sides of the CPA box for 15 min. The total time spent in the paired chamber and CPA scores were calculated for all groups.

To selectively measure nTS neuronal calcium transient activity, we injected an AAV containing a calcium sensor expressed under the synapsin promoter (pGP-AAV9-syn-jGCaMP8m-WPRE; Addgene viral prep # 162375-AAV9) into the nTS (500 nl per side; 0.3–0.5 mm anterior, ±0.4 mm lateral, and 0.4 mm ventral to brain surface, relative to calamus scriptorius). Rats were allowed at least 3 weeks to recover from nanoinjection surgery to allow for robust viral expression in the nTS before IV catheters were implanted as described above. On the day of the experiment, rats were deeply anesthetized with isoflurane (3–4% induction, 2% maintenance), placed in the stereotaxic apparatus, and the nTS was exposed as described above. To overcome limitations associated with chronic fiber implants in the nTS, a wireless photometry headstage (TeleFipho, Amuza Inc) was secured to a 5 mm flat-tipped silica optic fiber cannula (400 μm c.d./470 μm o.d., NA 0.37, 2.5 mm receptacle; Doric) and mounted the stereotaxic arm with an apparatus 3D printed in house to permit simultaneous nTS exposure and photometry recordings. The optic fiber was lowered to the site of the dorsal brainstem and firmly pressed on the surface above the nTS until a signal was detected (~0.3–0.5 mm ventral from the surface). An LED generated blue light was bandpass-filtered (445–490 nm) to excite jGCaMP8m and emission fluorescence was detected by an internal photodiode detector for green light (bandpass filtered at 500–550 nm). An internal DC amplifier transmitted the data (16-bit arbitrary unit; AU) to a wireless receiver (TeleFipho, Amuza Inc) and the data was extracted in real-time using TeleFipho software (Amuza Inc) at a sampling rate of 100 Hz. The offset was set to 90° and the LED power was adjusted to achieve an optimal signal output (30–40 k a.u.) not to exceed 36 mW power, and remained constant throughout the recording. Baseline activity of GCaMP fluorescence nTS-infected cells was measured after at least 5 min to mitigate effects of baseline drift in signal due to slow photobleaching artifacts. Then, baseline activity was measured for at least 30 s prior to administration of either fentanyl (20 µg/kg, i.v.) or NLXM (5 mg/kg, i.v.). After response activity was recorded, rats received a second intravenous injection of either an opioid receptor antagonist (NLX,1 mg/kg or NLXM, 5 mg/kg) or fentanyl (20 µg/kg), respectively. Each intravenous administration was manually time locked to calcium transient recordings by a second experimenter and response activity following each injection was recorded for 60 s before administration of the second injection or conclusion of the experiment. Rats were transcardially perfused with PFA at the end of the experiment as described above and viral expression was validated for all animals.

For analysis of calcium transient activity, custom MATLAB scripts (https://doi.org/10.5281/zenodo.15042135) were used as previously described (Higginbotham et al., 2025). The raw data were fit to a double-exponential curve and subtracted from the raw data to account for any residual effects of baseline drift. MATLAB Signal Analyzer (RRID:SCR_001622) was used to extract regions of interest (ROIs) in the signal in 90 s traces (30 s pre-treatment, 60 s post-treatment) and traces were aligned to the time of injection. ROIs were standardized as z-scores using the sample standard deviation, S, where S=∑i=1n(xi−X¯)2n−1 such that signals were centered to have a mean of 0 and scaled to have a standard deviation of 1. Based on this, the AUC of ROI z-scores were calculated in 30 s bins using the trapezoidal rule (trapz function in MATLAB) and one-sample t-tests were used to determine differences relative to baseline (theoretical mean = 0). For presentation, transients were down sampled by a factor of 10 with a phase offset of 2 and the resultant 10 Hz traces were smoothed using a moving mean duration of 5% of total ROI.

Immunohistochemical procedures were performed as previously described (Ruyle et al., 2019). Brainstem sections containing the nTS were sliced using a cryostat (Leica) at a thickness of 40 µm. Free-floating sections were rinsed in 0.01 M PBS, blocked in 10% Normal Donkey Serum (Sigma) in 0.01 M PBS-Triton, and then incubated with the following antibodies: cFos (Rabbit anti-cFos; ab190289, Abcam; RRID:AB_2737414); tyrosine hydroxylase (Mouse anti-TH; MAB318, Millipore; RRID:AB_2201528). The following day, sections were rinsed with 0.01 M PBS, incubated with Donkey anti-Rabbit Cy3 and Donkey anti-Mouse Alexa fluor 488 (1:200; in 3% NDS and 0.3% Triton in 0.01 M PBS) and used to visualize targets of interest (Jackson ImmunoResearch).

IR and viral expression were examined with a Leica DMR microscope at ×20 magnification. 20 µm thick z-stacks of the nTS (2 µm per plane) were collected. ImageJ (v. 1.48, NIH) software was used for post-processing and analysis. In all groups, three sections of the nTS (corresponding to –360, 0, +360 µm relative to calamus scriptorius) were collected.

Using ImageJ software (RRID:SCR_003070), the regions containing the nTS were outlined, and unilateral counts of Fos-IR, TH-IR, and co-labeled Fos- and TH-IR neurons were performed using a custom-made plugin (Cell Counter) for each of the 3 nTS sections per animal. Cell counts were performed by two individuals blinded to the treatment groups. The total number of each phenotype was summed from these three sections, and the percentage of TH-IR cells co-expressing Fos-IR was determined.

Statistical analyses were performed using GraphPad Prism v10.4.0. Data collection and analyses were performed blinded to the conditions of each experiment. For all experiments, the normality of sample data was determined using D’Agostino and Pearson tests and Shapiro–Wilk tests. Statistical significance was taken as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, as determined by two-way repeated measures ANOVA followed by Sidak’s post hoc test; one-way ANOVA with Tukey post hoc test, two-tailed unpaired t-test, two-tailed paired t-test, and one-sample t-test. Detailed statistical reporting for all results is available in the Source data files.