Dosing with formoterol induces periorbital allodynia

This experiment aimed to examine if formoterol induces headache-like periorbital allodynia behavior in mice and, if so, the degree to which this may occur. Formoterol (0.03 mg/kg, i.p.) or vehicle were administered daily for 7 or 42 days. Pre-drug baselines measurements were obtained prior to administration of the initiation dose of either formoterol or vehicle, after which experimental, post-drug values were measured weekly for up to 6 weeks. As hypothesized, there were no significant differences observed in the vehicle treated mice at any time point tested compared to baseline measurements (Fig. 1B; BL vs. vehicle, p > 0.05 at any time point, as assessed by two-way ANOVA with Tukey’s post-test, n = 6 in each group). However, in the formoterol treated mice there were persistent headache-like periorbital allodynic behaviors observed at almost all timepoints tested as soon as 7 days post drug administration (Fig. 1B; veh vs. form, 7 days: p = 0.0031, 14 days: p = 0.0113, 21 days: p = 0.0124, 28 days: p = 0.0055, 35 days: p = 0.0021, 42 days: p = 0.0072, as assessed by two-way ANOVA with Tukey’s post-test, n = 7 in each group; veh: mean ± SEM = 1.67 ± 0.04993, standard deviation = 0.1321; form: mean ± SEM = 0.8998 ± 0.1740, standard deviation = 0.4605). There were no significant differences in the formoterol treated mice between day 7 and day 42 (7-day vs 42-day: p = 0.5318, n = 6/timepoint), suggesting that while the functional outcome of the behavior does not change, the mechanism driving the behavior may be. These data suggest that formoterol does induce headache-like periorbital allodynic behaviors in the mice.

Fig. 1

Formoterol induces periorbital allodynia 7 days after drug initiation out through 42 days of treatment. 8-week-old female C57Bl/6J mice were acclimated to the assay chambers and Von Frey filaments 3 times prior to baseline measurements; experimental measurements were taken weekly post-drug initiation for 42-days, formoterol (0.03 mg/kg, i.p.) or vehicle were administered daily for 42 days. A Timeline of experimental setting. B Formoterol treated mice began to exhibit headache-like periorbital allodynic behavior 7-days post drug initiation as compared to vehicle treated and maintained this periorbital sensitivity throughout the 42-day testing period. Data represented as mean of threshold (g) ±SEM. (*denotes p\(\;\le\;\)0.05, **denotes p\(\;\le\;\)0.01, compared to vehicle treated mice)

Chronic administration of formoterol induces light sensitivity

As discussed above, a frequently occurring comorbidity in headache patients is the presence of photophobia. In the first experiment, naïve female mice were exposed to 6 variable lux between 23.8 and 735 lux in a light/dark box assay, this was used as pre-drug baseline. No significant sensitivity to light between any of the tested lux was observed (Fig. 2A; p > 0.05, as assessed by one-way ANOVA with Tukey’s post hoc, n = 9; mean ± SEM = 65.02 ± 3.307, SD = 8.100). Mice were baselined at 107.7 lux. There was no difference in time in light at this lux until days 35 and 42 compared to their baseline (Fig. 2B; veh vs. form, 35 days: p = 0.0130, 42 days: p = 0.0045; veh: mean ± SEM = 110.8 ± 10.70, SD = 28.30; form: mean ± SEM = 87.09 ± 4.567. SD = 12.08). At days 7 and 14 post-drug initiation there was a decrease in light aversion in both vehicle and formoterol treated mice, this trend was not observed at any following timepoints (Fig. 2C; veh vs. form, (1) 7 days: p = 0.0333; BL vs. veh, (2) 7 days: p < 0.001, (3) 14 days: p = 0.0382; BL vs. form, (4) 7 days: p = 0.03, (5) 14 days: p = 0.0026; veh: mean ± SEM = 0.1720 ± 0.0709, SD = 0.187; form: mean ± SEM = 0.225 ± 0.0462, SD = 0.122). Continuing with 107.7 lux, formoterol treated mice showed an increase in time spend in the dark chamber on days 28, 35 and 42 post-drug initiation compared to vehicle treated mice (Fig. 2D, veh vs. form, 28 days: p = 0.0496, 35 days: p = 0.0004, 42 days: p = 0.0066; veh: mean ± SEM = 128 ± 11.5, SD = 30.5; form: mean ± SEM = 146 ± 9.70, SD = 25.7).

When comparing aversion at different lux, sensitivity was not observed until the 28-day timepoint, at which point formoterol treated mice spent less time in the 23.8, 53.1, 107.7, 300.42 and 735 lux (Fig. 2E; veh vs. form, 23.8 lux: p = 0.0436, 53.1 lux: p = 0.0478, 107.7 lux: p = 0.0464, 300.42 lux: p = 0.0237, 735 lux: p = 0.0241; veh: mean ± SEM = 93.00 ± 1.610, SD = 3.944; form: mean ± SEM = 123.7 ± 1.599, SD = 3.918). Sensitivity continued to be observed at the 35-day timepoint at 107.7 and 735 lux (Fig. 2F; veh vs. form, 107.7 lux: p = 0.0064, 735 lux: p = 0.0238; veh: mean ± SEM = 100.5 ± 3.769, SD = 9.233; form: mean ± SEM = 129.4 ± 2.109, SD = 5.165). Lastly, treatment with formoterol for 42-days showed a decrease in time spent in the light at the 53.1, 107.7, 300.42 and 753 lux (Fig. 2G; veh vs. form, 53.1 lux: p = 0.0017, 107.7 lux: p = 0.0005, 300.42 lux: p = 0.0069, 735 lux: p = 0.00002; veh: mean ± SEM = 94.56 ± 4.534, SD = 10.64, SEM = 4.342; form: mean ± SEM = 131.3, SD = 11.11, SEM = 4.534).

All data was assessed by two-way ANOVA with Tukey’s post-test, with an n of 10 in each group, data for 7-21DPI can be found in supplemental Figure 1.

Fig. 2

Chronic administration of formoterol induces photophobia as assessed via the light/dark box assay. 8-week-old female C57Bl/6J mice were acclimated to the assay chambers twice for 30m prior to baseline measurements, experimental measurements were then taken weekly post drug initiation for 42-days. Mice were tested at each lux (23.8, 53.1, 107.7, 163, 300.42, and 735) for 5m per lux. At all lux tested naïve mice showed no difference in time spent in light (A). At the 107.7 lux, formoterol treated mice spent less time in the light at days 35 and 42 (B), the light aversion index shows moderate habituation ay days 7 and 14 for both the formoterol and vehicle treated mice (C). Formoterol treated mice spent more time in the dark at days 28, 35 and 42 than the vehicle treated mice (D). In the lux curve, starting 28 days post drug initiation (DPI), formoterol treated mice spent significantly less time in the light at 23.8, 53.1, 107.7, 300.42 and 735 lux as compared to vehicle control (E). 35 DPI, formoterol treated mice spent significantly less time in light at lux 107.7 and 735 (F) and at 42 DPI, formoterol treated mice spent significantly less time in the light at lux 53.1, 107.7, 300.42 and 753 as compared to vehicle control (G). Chronic exposure to formoterol does induce light avoidance behaviors starting after 28 days of drug application. Data represented as time in light (s) ±SEM (*denotes p\(\;\le\;\)0.05, **denotes p\(\;\le\;\)0.01, ***denotes p\(\;\le\;\)0.001 compared to vehicle treated mice)

Formoterol does not induce anxiety-like behaviors

Anxiety is a common side-effect of formoterol as reported by the FDA, and a co-morbidity of chronic headache disorders [30, 58,59,60]. The elevated plus maze was used to assess anxiety like behavior with formoterol treatment. There was no difference observed between the formoterol-treated groups and the vehicle-treated groups in the closed arm (Fig. 3A; veh vs. form, p > 0.05 as assessed by two-way ANOVA with Tukey’s post-test, n = 10 in each group; veh: mean ± SEM = 1.569 ± 0.04470, SD = 0.1095; form: mean ± SEM = 1.464 ± 0.02862, SD = 0.07011). Formoterol treated mice spent more time in the open arm than vehicle treated mice at 14 days, 35 days and 42 days (Fig. 3B; veh vs. form, 14 days: p = 0.037, 35 days: p = 0.0131, 42 days: p = 0.0268 as assessed by two-way ANOVA with Tukey’s post-test, n = 10 in each group; veh: mean ± SEM = 0.3214 ± 0.03066, SD = 0.07509, SEM = 0.03066; form: mean ± SEM = 0.4986 ± 0.01874, SD = 0.04590). While individuals taking formoterol have reported experiencing anxiety, this is not reported in all patients. Despite these clinical observations, the presence of anxiety-like behavior was not observed in our animal model, the anxiety associated with formoterol treatment could remain patient-relevant.

Fig. 3

Anxiety-like behaviors are not observed under long-term treatment with formoterol. 8-week-old female C57Bl/6J mice were baselined for 300s/test prior to drug initiation; experimental measurements were taken weekly post-drug initiation for 42-days, formoterol (0.03 mg/kg, i.p.) or vehicle were administered daily for 42 days. A Throughout the 42-day study no difference between treatment groups was observed in terms of time spent in the closed arm. B Formoterol treated mice spent more time in the open arm at days 14, 35, and 42. Data represented as time spent in arm/baseline ± SEM. (*denotes p\(\;\le\;\)0.05, compared to vehicle control)

Age and formoterol treatment dynamically regulates levels of endocannabinoid lipids, 2-AG and AEA, and CB1R expression within the PAG in a time-dependent manner

Previous work suggests that levels of 2-AG are reduced within the PAG, but not the TG or Vc, during cortical spreading depression-associated allodynia and MOH models using sumatriptan and morphine [25, 26]. Given the duration of the study, the first experiment investigated potential age-related differences within PAG with respect eCB levels and CB1 receptor expression in naïve mice; the following ages were assessed: 9-weeks-old, to model the 7-day time point, and 15-weeks-old, to model the 42-day duration. AEA lipid levels were higher in the 15-week-old mice as compared to the 9-week-old mice. In contrast, 2-AG lipid levels were lower in the 15-week-old mice as compared to the 9-week-old mice (Fig. 4A; 9-week vs 15-week, AEA: p < 0.0001, 2-AG: p = 0.0479, as assessed by unpaired t-test, n = 8–9; AEA: 9w: mean ± SEM = 0.02424, SD = 0.006535, SEM = 0.002311; 15w: mean ± SEM = 0.05787, SD = 0.01334, SEM = 0.004445; 2-AG: 9w: mean ± SEM = 19.37, SD = 7.149, SEM = 2.527; 15w: mean ± SEM = 13.90, SD = 2.549, SEM = 0.8495). Additionally, protein expression of CB1R was significantly higher in the 15-week-old mice versus the 9-week-old mice (Fig. 4B; 9-week vs 15-week, p = 0.0033 as assessed by unpaired t-test, n = 4–5; 9w: mean ± SEM = 0.7019, SD = 0.1906, SEM = 0.09530; 15w: mean ± SEM = 1.161, SD = 0.1253, SEM = 0.05605).

These results indicate that rigorous assessment of eCB levels and CB1R protein expression in formoterol or vehicle treated mice required results be normalized to their specific age-matched naive controls. Mice were treated for either 7- or 42-days with formoterol or vehicle starting at 8-weeks of age. After 7-days of formoterol treatment, there was a significant decrease in levels of AEA within the PAG versus the vehicle treated mice; after 42-days of formoterol treatment, there was a significant increase in levels of AEA versus the vehicle treated mice (Fig. 4C; veh vs. form, 7 days: p = 0.0083 n = 8, 42 days: p = 0.0010 n = 12–14 as assessed by unpaired t-test; 7d AEA: veh: mean ± SEM = 102.4, SD = 28.60, SEM = 10.11; form: mean ± SEM = 66.57, SD = 16.54, SEM = 5.847; 42d AEA: veh: mean ± SEM = 47.45, SD = 16.92, SEM = 4.523; 42d: mean ± SEM = 76.04, SD = 21.87, SEM = 6.313). Regarding 2-AG, after 7-days of formoterol mice exhibited a decrease in levels of 2-AG within the PAG as compared to vehicle control; however, this reduction of 2-AG level was not observed at the 42-day timepoint (Fig. 4D; veh vs. form, 7 days: p = 0.0151 n = 8, 42 days: p > 0.05 as assessed by unpaired t-test; 7d 2-AG: veh: mean ± SEM = 159.3, SD = 62.17, SEM = 21.98; form: mean ± SEM = 94.11, SD = 23.94, SEM = 8.463; 2-AG: veh: mean ± SEM = 73.30, SD = 36.10, SEM = 9.649; form: mean = 81.62, SD = 32.74, SEM = 9.451). At both the 7-day and 42-day timepoints there was a decrease in CB1R protein expression in the formoterol treated mice versus the vehicle treated mice (Fig. 4E; veh vs. form, 7 days: p = 0.0042 n = 4 per group, 42 days: p = 0.0033 n = 9–10 per group as assessed by unpaired t-test; 7d: veh: mean ± SEM = 163.7, SD = 9.603, SEM = 4.802; form: mean ± SEM = 120.9, SD = 16.52, SEM = 8.261; 42d: veh: mean ± SEM = 138.2, SD = 16.91, SEM = 5.346; form: mean ± SEM = 114.3, SD = 13.24, SEM = 4.414). Whole blot representations are in Supplemental Figures 2 and 3, raw densitometry values can be found in Supplemental Figure 4. All together, these results suggest that treatment with formoterol dynamically regulates eCB tone within the PAG.

Fig. 4

AEA, 2-AG, and CB1R expression in the PAG changes with age and with formoterol treatment. The PAGs from C57Bl/6J female age-matched naïve mice were harvested and eCB lipids assessed via LC-MS, (A) dynamic age-related changes were observed for both AEA and 2-AG. Data represented as mean ± SEM in pmol/mg unit. B In the naïve mice, protein expression of cannabinoid receptor 1 (R) changes with age. Data represented as mean ± SEM. β-actin was used as loading control. C 7d post drug initiation, AEA levels are significantly decreased in formoterol treated mice versus vehicle treated mice but at 6w post drug initiation AEA levels are significantly increased in formoterol treated mice. D 2-AG level within the PAG is significantly decreased in formoterol treated mice versus vehicle treated mice at 7-days, but by 42-days there is no difference observed between experimental groups. E expression within PAG in formoterol treated mice is significantly decreased as compared to vehicle treated mice both 7d and 6w post drug initiation. Data represented as percentage of naïve ± SEM. (*denotes p\(\;\le\;\)0.05, **denotes p\(\;\le\;\)0.01, ***denotes p\(\;\le\;\)0.001, **** denotes p\(\;\le\;\)0.0001 compared to age-matched or vehicle treated mice)

ADRB2 receptor expression does not change with age but is altered by chronic treatment with formoterol

The protein expression of the ADRB2 within the PAG region, the target receptor of formoterol was assessed by Western immunoblotting. The ADRB2 expression was not dynamically regulated with age in PAG tissue (Fig. 5A; 9-week vs 15-week, p > 0.05, as assessed by unpaired t-test, n = 4–5; 9w: mean ± SEM = 0.6749, SD = 0.08414, SEM = 0.04207; 15w: mean ± SEM = 0.7202, SD = 0.09640, SEM = 0.04311). There was a significant increase in protein expression at the 7-day timepoint, which was diminished by the 42-day timepoint (Fig. 5B; veh vs. form, 7 days: p = 0.0227, 42 days: p > 0.05, as assessed by unpaired t-test, 7 days n = 4 per group, 42 days n = 9–10; 7d: veh: mean ± SEM = 175.2, SD = 34.65, SEM = 17.32; form: mean ± SEM = 256.6, SD = 40.60, SEM = 20.30; 42d: veh: mean ± SEM = 69.91, SD = 12.90, SEM = 4.080; form: mean ± SEM = 73.49, SD = 15.37, SEM = 5.123). This effect was not unexpected, as repeated agonism of a G protein-coupled receptor can initially cause an increase in receptor expression, followed by a decrease due to desensitization of the receptor [61]. Whole blot representations are in Supplemental Figures 2 and 3, raw densitometry values can be found in Supplemental Figure 4.

Fig. 5

Formoterol treatment induced changes in the expression of ADRB2 receptor within the PAG 7-days post-initiation. The PAGs from C57Bl/6J female age-matched naïve mice were harvested and ADRB2 receptor protein expression assessed by Western immunoblotting, (A) age-related changes were not observed. Data represented as mean ± SEM. β-actin was used as loading control. B At 7 DPI, there is a significant increase in the expression of ADRB2 receptor in the formoterol treated mice versus the vehicle treated mice. No significant difference was observed in the expression of ADRB2 receptor 42 DPI, compared to vehicle control. Data represented as percentage of naïve ± SEM. β-actin was used as loading control. (*denotes p\(\;\le\;\)0.05 compared to age-matched or vehicle treated mice)

Targeting of the endocannabinoid system reverses the observed formoterol-induced periorbital allodynia and alters levels of AEA

Given that endocannabinoid lipid tone (2-AG and AEA) and CB1R expression were reduced on Day 7 and that AEA was increased over controls on Day 42 of formoterol-induced headache (Fig. 4), the next study asked if normalizing eCB tone using pharmacological inhibitors of 2-AG degradation, MAGL (MJN) and AEA degradation, FAAH (JNJ) or a non-selective CB1/CB2R agonist (WIN55,212-2), could mitigate the behaviors at these time points after a single bolus. All mice received formoterol daily for 7- or 42-days with periorbital allodynia behavior assessed prior to drug administration. On the final day of formoterol administration (D67 or D42), periorbital allodynia was reassessed prior to inhibitor/agonist injection and every 30-minutes post injection (PI) for 4-hours. Prior to drug administration, all three groups showed periorbital sensitivity compared to the baseline measurements (Fig. 6A; baseline vs. all treated groups, PI 7d BL: p < 0.001; Veh BL: mean ± SEM = 1.666, SD = 0.4219, SEM = 0.1594, Veh 7d BL: mean ± SEM = 0.6514, SD = 0.5737, SEM = 0.2168, MJN/JNJ BL: mean ± SEM = 1.962, SD = 0.09390, SEM = 0.03833, MJN/JNJ 7d, BL: mean ± SEM = 0.4517, SD = 0.3301, SEM = 0.1348; WIN BL: mean ± SEM = 1.918, SD = 0.2009, SEM = 0.08200, WIN 7d BL: mean ± SEM = 0.6600, SD = 0.3327, SEM = 0.1358). 30-minutes after drug application, reversal of formoterol-induced periorbital allodynia was observed in both treatment groups, compared to vehicle control (form + veh vs. form + MJN/JNJ, 30-minutes PI: p = 0.0020, MJN/JNJ 7d BL: mean ± SEM = 0.4517, SD = 0.3301, SEM = 0.1348, MJN/JNJ 30m: mean ± SEM = 1.088, SD = 0.6590, SEM = 0.2691; WIN 7d BL: mean ± SEM = 0.6600, SD = 0.3327, SEM = 0.1358, WIN 30m: mean ± SEM = 1.088, SD = 0.6590, SEM = 0.2691; 1-hours through 4-hours PI: p < 0.0001; form + veh vs. form + WIN, 30-minutes through 4-hours PI: p < 0.0001; veh: mean ± SEM = 0.7324, SD = 0.3463, SEM = 0.1095; MJN/JNJ: mean ± SEM = 1.645, SD = 0.4830, SEM = 0.1527; WIN: mean ± SEM = 1.654, SD = 0.4340, SEM = 0.1372). All data was assessed by two-way ANOVA with Tukey’s post-test, with an n OF 8 in each group.

Levels of AEA and 2-AG within the PAG were then assessed at both timepoints described above. At the 7-day timepoint, there was a significant increase in levels of AEA in the MJN/JNJ treated mice compared to vehicle control (Fig. 6B; form + veh vs. form + MJN/JNJ, p < 0.0001, as assessed by one-way ANOVA with Tukey’s posttest, n = 7–8 per group; veh: mean ± SEM = 83.51, SD = 20.63, SEM = 7.796; MJN/JNJ: mean ± SEM = 133.8, SD = 15.88, SEM = 5.614). No significant changes in 2-AG level were detected in the MJN/JNJ treated group compared to vehicle control (Fig. 6C; form + veh vs. form + MJN/JNJ, p > 0.05, as assessed by one-way ANOVA with Tukey’s posttest, n = 7–8 per group; veh: mean ± SEM = 82.62, SD = 96.04, SEM = 36.30; MJN/JNJ: mean ± SEM = 111.8, SD = 152.3, SEM = 53.84;). The WIN55,212 treatment did not induce significant changes in either endocannabinoid level at day 7 compared to vehicle control (Fig. 6B and C; form + veh vs. form + WIN, 7d PI: p > 0.05 as assessed by one-way ANOVA, n = 7-8per group; AEA: mean ± SEM = 102.2, SD = 16.64, SEM = 5.882; 2-AG: mean ± SEM = 125.1, SD = 105.4, SEM = 37.27).

At the 42-day timepoint when CB1R expression remained downregulated, a separate cohort of mice were given vehicle or WIN55,212 and behavior was assessed as above. Only WIN55,212 was administered at this time based on the LC-MS and WB data, showing an increase in the levels of AEA and no difference in the levels of 2-AG in the formoterol treated mice, suggesting the degradation enzymes MAGL and FAAH are not playing major roles in headache-like periorbital allodynic behavior at this later timepoint (Fig. 4). At day 42, mice exhibited periorbital allodynia as compared to pre-drug baseline (Fig. 6D; baseline vs. all treated groups, PI 42d BL: p < 0.0001; BL: mean ± SEM = 1.733, SD = 0.3840, SEM = 0.1358, form + Veh 7d BL: mean ± SEM = 0.1163, SD = 0.1602, SEM = 0.05663, BL: mean ± SEM = 1.747, SD = 0.3501, SEM = 0.1238, form + WIN 42d BL: mean = 0.1163, SD = 0.1113, SEM = 0.03937). Vehicle administration did not alter facial withdrawal thresholds at any time point evaluated. In contrast, 30 minutes post administration of WIN55,212, periorbital allodynia was significantly reversed, and this maintained throughout the four hour test period (Fig. 6D; form + veh vs. form + WIN, 30-minutes through 4-hours PI: p < 0.0001; veh: mean ± SEM = 0.3735, SD = 0.4886, SEM = 0.1545, WIN: mean ± SEM = 1.494, SD = 0.5646, SEM = 0.1785). All data was assessed by two-way ANOVA with Tukey’s post-test, with an n of 8 in each group.

At the 42-day timepoint there was an increase in levels of AEA observed in the WIN treated mice versus the vehicle treated mice (Fig. 7E; form + veh vs. form + WIN, 42d PI: p = 0.0018 as assessed by unpaired t-test, n = 8 per group; veh: mean ± SEM = 86.56, SD = 13.56, SEM = 4.796, WIN: mean ± SEM = 107.9, SD = 8.027, SEM = 2.838). There was also no difference in levels of 2-AG in the WIN treated mice versus vehicle control (Fig. 6F; form + veh vs. form + WIN, 42d PI: p > 0.05 as assessed by unpaired t-test, n = 8 per group; veh: mean ± SEM = 42.87, SD = 16.37, SEM = 5.787, WIN: mean ± SEM = 45.77, SD = 10.23, SEM = 3.618). These data suggest that the endocannabinoid system plays a role in formoterol-induced headache-like periorbital allodynic behaviors, supporting the hypotheses that there is an interaction occurring between the endocannabinoid and adrenergic system within PAG.

Fig. 6

MAGL/FAAH and CB1R inhibitors reversed formoterol induced periorbital allodynia. 8-week-old female C57Bl/6J mice were acclimated to the assay chambers and Von Frey filaments 3 times prior to baseline measurements, followed by treatment with formoterol for 7 days or 42 days. Mice given formoterol for 7 days received either MJN/JNJ, WIN,55,212-2, or vehicle on the last day of formoterol administration. A At time of baseline, mice did not exhibit headache-like periorbital allodynic behaviors; after receiving formoterol for 7 days, increase in periorbital sensitivity was observed. A 30-minutes after administration of MJN/JNJ or WIN, a significant decrease in headache-like periorbital allodynic behavior was observed, with no significant difference in threshold values between day 0 baseline and 1 hour post inhibitor administration for these treatments. B MJN/JNJ administration induced significant increase in the level of AEA within the PAG compared to vehicle control. C There are no significant changes observed in the levels of 2-AG in either treatment group compared to vehicle control. A second cohort of mice received formoterol for 6 weeks and were then given either WIN or vehicle. PAG was harvested 5 hours later. D In the 6w study, 30 minutes after administration of WIN 55,212, a significant decrease in headache-like periorbital allodynic behaviors was observed but values did not reach pre-drug administration baseline values until 2.5 hours post-treatment with WIN55,212. In a separate set, mice received formoterol daily for 7 days, at which point they received either MJN/JNJ, WIN, or vehicle. PAG was harvested after 5 hours and subjected to LC-MS to measure endocannabinoid levels. E WIN treated mice exhibited an increase in levels of AEA versus vehicle treated mice, and (F) levels of 2-AG were not significantly different between the two groups. Data represented as threshold (g) ± SEM or percentage of naive ± SEM. (### denotes p\(\;\le\;\)0.001 compared to Day 0 BL; **denotes p\(\;\le\;\)0.01, **** denotes p\(\;\le\;\)0. compared to form+veh)