Sleep restriction during opioid abstinence affects the hypothalamic-pituitary-adrenal (HPA) axis in male and female rats

Figures & data

Figure 1. Experimental protocol timeline. Rats were allotted 7 d for recovery from surgery and 8 d for baseline measurements, which included diurnal and restraint stress ACTH and corticosterone. After 10 d of sucrose SA and 10 d of oxycodone SA during a 26-d timespan, rats entered the abstinence phase and were acclimated to the sleep restriction apparatuses. During the next 35 d of abstinence, sleep restriction or ambulation control conditions were carried out. HPA axis assessments were repeated on Days 20–23 of sleep restriction or control conditions. The final nine experimental days of sleep restriction or control conditions were composed of extinction testing, footshock reinstatement, and tissue harvests.

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Protocol: HPA dynamics in rats before and during abstinence from opioids; during the abstinence period, rats underwent a sleep restriction protocol or were ambulatory controls.

Table 1. mRNA data not shown in Figure 4 (fold-change from Male AC).

	Male		Female
	AC	SR	AC	SR
Hypothalamus (PVN)
Gilz	1.00 ± 0.08	0.63 ± 0.05^a	0.73 ± 0.03^b	0.62 ± 0.03
Per1	1.00 ± 0.11	0.58 ± 0.03^a	0.63 ± 0.03^b	0.71 ± 0.06
Nr3c1	1.00 ± 0.04	0.91 ± 0.04	0.91 ± 0.03	0.88 ± 0.03
Nr3c2	1.00 ± 0.03	0.89 ± 0.02	0.95 ± 0.02	0.95 ± 0.04
Anterior pituitary
Avpr1a	1.00 ± 0.10	0.87 ± 0.06	1.12 ± 0.18	1.00 ± 0.11
Avpr1b	1.00 ± 0.05	0.87 ± 0.04	0.49 ± 0.06^b	0.42 ± 0.04^b
Crhbp	1.00 ± 0.06	1.31 ± 0.15	1.12 ± 0.17	1.00 ± 0.12
Crhr1	1.00 ± 0.04	0.94 ± 0.04	0.54 ± 0.05^b	0.57 ± 0.07^b
Crhr2	1.00 ± 0.05	0.89 ± 0.06	0.39 ± 0.08^b	0.50 ± 0.09^b
Fkbp5	1.00 ± 0.13	0.96 ± 0.07	0.52 ± 0.05^b	0.42 ± 0.06^b
Gilz	1.00 ± 0.08	0.93 ± 0.05	0.71 ± 0.06^b	0.59 ± 0.06^b
Nr3c1	1.00 ± 0.07	0.97 ± 0.04	0.57 ± 0.06^b	0.49 ± 0.05^b
Nr3c2	1.00 ± 0.04	0.96 ± 0.03	0.44 ± 0.05^b	0.40 ± 0.04^b
Per1	1.00 ± 0.16	0.96 ± 0.05	0.77 ± 0.10	0.85 ± 0.05
Pomc	1.00 ± 0.09	1.11 ± 0.09	0.55 ± 0.08^b	0.58 ± 0.07^b
Adrenal cortex
Cyp11a1	1.00 ± 0.10	0.91 ± 0.25	1.41 ± 0.05^b	1.29 ± 0.10^b
Ldlr	1.00 ± 0.32	1.12 ± 0.23	1.29 ± 0.19	0.91 ± 0.12
Mc2r	1.00 ± 0.08	1.04 ± 0.07	0.80 ± 0.03^b	0.90 ± 0.06^b
Star	1.00 ± 0.12	1.07 ± 0.13	1.36 ± 0.13^b	1.19 ± 0.10

^aDifferent from AC within sex. ^bDifferent from male within AC or SR. Bolded rows are highlighted in the results sections. Other findings are reported for completeness.

Figure 2. Diurnal plasma ACTH (upper panels) and corticosterone (lower panels) in female and male rats Before and then During abstinence from opioids in rats under sleep restricted (SR) conditions vs. ambulatory controls (AC) (Male: N = 15–16 per mean/SE for treatment groups; Female: N = 14–16 per mean/SE for treatment groups). All statistical symbols are p < 0.05. (a) Male different from female within Before or During, (b) During different from Before within sex. For overall ANOVA and all specific comparisons, see the link to Supplementary Table 2 in the reference list (Raff, 2022b).

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Opioid abstinence resulted in an increase AM plasma ACTH and corticosterone in male but not female rats regardless of sleep state (left panels).

Figure 3. Plasma ACTH and corticosterone response to 30 min of restraint in male and female rats Before and then During abstinence from opioids in ambulatory control rats and rats under sleep restricted conditions (N values as in Figure 2; mean/SE). All statistical symbols are p < 0.05. Symbol colors are specific to AC (blue) or SR (red) rats. (a) Different from 0 min; (b) above the dashed line indicates an overall difference between AC and SR “During” by Hotelling’s test (p < 0.019); (c) different from “Before” at that time point. c above the dashed line indicates an overall difference from “Before” by Hotelling’s test for AC (p = 0.0027) and SR (p = 0.026) rats. (d) Different from male within same treatment at same time point. For overall ANOVA and all specific comparisons, see link to Supplementary Table 2 in the reference list (Raff, 2022b).

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The corticosterone response to restraint was attenuated in sleep restricted male rats during abstinence compared to before abstinence (bottom panels). Opioid abstinence attenuated the ACTH response to restraint in female rats (top right panel). Sleep restricted female rats did not achieve the full restraint recovery nadir in corticosterone (bottom right panel).

Figure 4. Paraventricular nucleus mRNAs for corticotropin-releasing factor (CRH) and arginine vasopressin (AVP) from brains obtained at the end of the abstinence/sleep restriction vs. abstinence/ambulatory control periods. Data shown as fold changes (calculations described in Methods section). N = 6 per mean/standard error. All statistical symbols are p < 0.05. (a) Different from AC within sex. (b) different from male within treatment group.

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Hypothalamic PVN Crh (left panel) and Avp mRNA (right panel) expression was increased in male but not female sleep restricted rats during abstinence.

Figure 5. Plasma copeptin concentration at the end of the abstinence/sleep restriction vs. abstinence/ambulatory control periods (N = 9 per mean/SE). (a) Different from AC within sex (p < 0.001). (b) Different from male within treatment group (p < 0.001).

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Plasma copeptin was increased in male but not female sleep restricted rats during abstinence.

Raff, H. (2022b). Supplementary_Table 2_Statistics_PDF.pdf. Deposited 11-25-2022 FigShare2022. https://figshare.com/articles/dataset/Raff_Opioid_Restraint_ANOVA_Supplementary_Table_2/21623355

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Data availability statement

All data generated or analyzed during this study are included in this published article or in the supplementary files in the data repository cited in the references.