Fig. 1 Postmortem brain tissue from heroin addicts shows an increased number of hypocretin-producing neurons. Subject characteristics are presented in Table 1. (A) Immunohistochemistry showed that there was a 54% increase in the number of detectable hypocretin neurons in hypothalamic brain tissue from human heroin addicts (n = 5) relative to hypothalamic tissue from human control subjects [n = 7; P = 0.0009, t = 8.89, df = 10 (t test)]. (B) Immunohistochemical staining of postmortem brain tissue showed that hypocretin cells were 22% smaller in brain tissue from heroin addicts compared to control subjects [P < 0.01, t = 2.78, df = 10 (t test)]. (C) Neurolucida mapping illustrates the distribution and increased number of hypocretin cells in brain tissue from heroin addicts relative to control subjects. Representative counts are given at three anterior-posterior positions: OT, optic tract; MM, mammillary bodies; Fx, fornix. (D) A representative example of immunohistochemical labeling of hypocretin cells in brain tissue from control individuals and heroin addicts is shown. Scale bar, 50 μm. (E) Left: Immunohistochemical staining showing that about 90% of hypocretin neurons in human brain tissue from control individuals also contained the neuropeptide Narp. This percentage did not significantly differ in brain tissue from human heroin addicts. Right: Representative images of immunohistochemical double staining for hypocretin (Hcrt) and Narp in hypothalamus of brain tissue from a heroin addict. ***P < 0.001, **P < 0.01, t test, compared to control.
Fig. 2 Dose-dependent effects of morphine administration on hypocretin cells in mouse brain. We administered morphine (blue) or saline (green) to wild-type littermate mice and then stained brain tissue for hypocretin-producing cells. (A) A fixed morphine dose (FD) of 100 mg/kg for 7 days, or an escalating morphine dose (ED) starting at 100 mg/kg for 3 days and increasing by 20% every third day for 7 days, did not significantly increase hypocretin cell number. However, a fixed dose of morphine (100 mg/kg) for 14 days and an escalating dose for 14 days (with a final dose of 180 mg/kg) both increased hypocretin cell number [P = 0.01, t = 4.23, df = 4 and +22%, P = 0.01, t = 4.52, df = 4 (t test), respectively). (B) Morphine doses of 10 mg/kg or higher all produced a significantly elevated number of hypocretin neurons compared to saline-injected mice. The elevation in hypocretin cell number at 50 mg/kg was 38%. Doses above 50 mg/kg produced no further increase [F7,16 = 8.1, P < 0.001 (ANOVA)]. (C) Shown are the effects of long-term (60 days) daily administration of morphine at doses of 10, 25, or 50 mg/kg. All three doses produced significant increases in hypocretin cell number as a percentage of control, with the largest increase (+26%) observed at a dose of 25 mg/kg [F3,12 = 13.5, P < 0.001 (ANOVA)]. (D) Shown is the mediolateral distribution of increased hypocretin cell number after 14 days of morphine administration at 50 mg/kg in wild-type mice. The elevation of hypocretin cell number was largest in the LH, but the effect was also significant in the MH [LH: P = 0.001, t = 11.94, df = 7; MH: P = 0.05, t = 2.44, df = 7 (t test)]. PFA, perifornical area. (E) Graph shows the persistence of the elevation of the number of hypocretin neurons after the termination of morphine administration to wild-type mice compared to mice injected with saline (control). Shown is the duration of hypocretin’s effects after termination of daily morphine (50 mg/kg) administration starting with the day after the final injection (day 0 of withdrawal) relative to control. After 14 days of morphine administration (top), hypocretin cell number remained significantly elevated for 4 weeks [F5,22 = 9.4, P < 0.001 (ANOVA)]. After 60 days of morphine administration (bottom), the significant elevation of hypocretin cell number lasted for 2 weeks [F5,22 = 6.3, P < 0.001 (ANOVA)]. (F) A 12.8 ± 2.8% decrease in hypocretin cell size was observed after administration of morphine (50 mg/kg) for 14 days to wild-type mice [significant interaction of treatment and withdrawal (F5,58 = 5.3, P < 0.0005)]. After 4 weeks of morphine withdrawal, hypocretin cell size returned to the size seen in saline-treated animals. (G) Representative neurolucida plots and photomicrographs illustrate the increased number and the reduced size of hypocretin cells after 2 weeks of morphine administration at 50 mg/kg fixed dose. Numbers indicate cell counts in section. Scale bars, 150 μm (neurolucida plots) and 50 μm (photomicrographs). 3V, third ventricle. (H) No change was observed in the percentage of cells double-labeled for both hypocretin and Narp in wild-type mice treated with morphine (50 mg/kg) for 14 days compared to control mice injected with saline. (I) We measured the effect of morphine pellet implantation on hypocretin cell number and size compared to control pellets in wild-type mice 72 hours after implantation. On average, cell size was decreased by 23% [P = 0.001 (Mann-Whitney U test), U = 5239]. n = 128 cells for control saline-injected mouse brain (green), and n = 149 cells for morphine-injected mouse brain (blue). (J) Shown are the effects of replacement of depleted morphine or control pellets at 3-day intervals for up to 7 or 14 days to mimic continuous opiate administration. (K and L) In contrast to hypocretin, MCH cell number and size were not affected by administration of morphine at 50 or 100 mg/kg for 14 days to wild-type mice. Dark green color in (K) and (L) distinguishes MCH neuron comparisons with saline control from hypocretin neuron comparisons with saline control. There was no change in MCH cell size compared to saline control. *P < 0.05, **P < 0.01, ***P < 0.001, t test with Bonferroni correction compared to saline control.
Fig. 3 Effect of morphine administration on preprohypocretin, preprodynorphin, Narp, and MCH expression in mouse brain. An escalating dose of morphine, starting at 100 mg/kg, was given for 14 days to wild-type mice. qPCR was performed to measure mRNA expression (A to C), and Western blotting was used to assess the amount of neuropeptide (D and E). (A to C) An increase in mRNA for preprohypocretin, Narp, and preprodynorphin was observed [preprohypocretin: P = 0.03, t = 2.99, df = 5; Narp: P = 0.02, t = 3.36, df = 5 (t test); preprodynorphin: P = 0.01, t = 3.65 df = 5 (t test)]. (D) Western blotting showed that 14 days of administration of morphine to wild-type mice increased preprohypocretin by 79% [P = 0.05, t = 2.51, df = 6 in each group (t test)] and returned to baseline within 2 weeks. (E) There was no significant change in MCH after 14 days of morphine administration. The difference remained nonsignificant from control after morphine withdrawal. WD, 2-week withdrawal. *P < 0.05, **P < 0.01, t test, compared to saline control.
Fig. 4 The increase in hypocretin cell number after morphine administration was not due to neurogenesis. (A) Morphine administration at 100 mg/kg for 14 days resulted in labeling of hypocretin cells in wild-type (WT) mice, but not in hypocretin knockout (KO) mice, indicating that labeling required the presence of hypocretin in neurons. Scale bar, 200 μm. (B) The increased number of hypocretin neurons after morphine administration was not due to neurogenesis. BrdU labeling to identify new neurons showed no significant increase in the number of new hypocretin neurons after 14 days of morphine (100 mg/kg) treatment of wild-type mice compared to control mice given saline injections. Right: BrdU-labeled cells in the perifornical area of saline-treated (top) and morphine-treated (bottom) animals. Scale bars, 100 and 40 μm. (C) Shown is immunohistochemical staining in the hypothalamus for doublecortin after 14 days of morphine (100 mg/kg) treatment of wild-type mice. Left: Representative photomicrograph shows the absence of doublecortin staining in the hypothalamus of a morphine-treated animal. Right: Representative photomicrograph shows normal doublecortin staining of dentate gyrus (DG) of the same animal. Scale bar, 100 μm.
Fig. 5 Effect of morphine administration on hypocretin cell activity in rats in vivo. A species-appropriate dose of morphine (15 mg/kg) injected into three freely moving rats resulted in an elevated neuronal discharge rate lasting for 3 hours accompanied by an increase in EMG activity. (A) Sleep rates are averages of mean rate determined by five 10-s samples in each of five hypocretin neurons from three rats in each sleep state: active waking, quiet waking, non-REM sleep, and REM sleep. Post-morphine injection rate was based on five 10-s samples in each neuron taken 15 min after morphine injection. One-way ANOVA of hypocretin neurons (n = 5): F4,16 = 18.2, P < 0.0001. Post hoc comparisons with Tukey/Kramer procedure: active waking/quiet waking, P < 0.05; active waking/non-REM sleep, P < 0.01; active waking/REM, P < 0.01. (B) Discharge rate of rat hypocretin neurons after morphine administration. Bottom: Traces show EEG activation immediately after morphine injection (left) and 3 hours after injection (right). Increased rate of discharge in hypocretin neurons lasted for 3 or more hours after injection of morphine. Expanded traces show the characteristic long average waveform of hypocretin neurons. (C) Histology showing three recording sites of hypocretin neurons in rat brain, labeled with arrowheads. Scale bar, 150 μm. #P < 0.05, ##P < 0.01, compared to active waking; **P < 0.0001 compared to quiet waking, non-REM sleep, and REM sleep.
Fig. 6 Reversal of hypocretin cell loss and cataplexy in narcoleptic mice after morphine administration. (A) The dashed horizontal red line shows the number of hypocretin cells in the brains of narcoleptic DTA mice maintained throughout the experiment on DOX, with 14 days of daily saline injections, followed by sacrifice. The green bar shows the number of hypocretin cells in the brains of DTA mice after DOX withdrawal for 1.5 days, followed by restoration of DOX administration and saline injections for 14 days. A 30% reduction of hypocretin cells relative to control was observed. When daily morphine (100 mg/kg) injections were given instead of saline for 14 days, the number of detected hypocretin cells was restored to baseline (blue bar). This difference was significant [***P = 0.003, t = 6.31, df = 4 (t test)]. Photomicrographs on the right show representative examples of hypocretin labeling in brains of DTA mice treated with saline (top) or morphine (bottom). Scale bar, 200 μm. (B) Morphine administration (blue) to DTA mice decreased cataplexy after 1 and 2 weeks of administration relative to control DTA mice receiving saline injections (green). Treatment effect: F1,6 = 148.4, P = 0.0001 (ANOVA). Changes after saline administration were not significant. Post hoc comparisons with Bonferroni correction revealed a significant difference at P < 0.01 between the saline-treated control DTA mice and the morphine-treated DTA mice at both 1 and 2 weeks of treatment. (C) Immunohistochemical staining shows that brain tissue from a human narcoleptic patient with cataplexy (01064, blue) treated for a long period with morphine has a higher number of hypocretin cells than does a case control narcoleptic patient with cataplexy (08023, yellow) not treated with morphine. The hypocretin cell counts in brain tissue from three control patients without narcolepsy or other identified neurological disorders are shown in green. The patients’ brains were willed to the Netherlands Brain Bank and preserved and analyzed using the same techniques (Table 1).
- Table 1 Characteristics of addicts, narcoleptic patients, and control subjects. PMI, postmortem interval; avg, average; MI, myocardial infarction; N/A, not available; SLE, systemic lupus erythematosus.
Subject Sex Age Cause of death Medications PMI pH Years in fix Estimated hypocretin cell no. Addicts (A) A-1 M 38 Heroin intoxication Oxycodone abuse, smoked 1 1/2 packs per day 20.0 6.84 3.5 121,173 A-2 F 26 Asthma complicating Narc intoxication Heroin, cocaine, methadone (110 g) for 8 months, nonsmoker 4.0 6.63 3.5 130,719 A-3 M 72 Cancer Former heroin addict, morphine alcohol “comfort” 4 days before death 19.0 3.6 111,466 A-4 M 28 Methadone intoxication and cocaine use Heroin, cocaine, methadone 13.0 5.52 3 123,810 A-5 F 19 Methadone intoxication High-dose drug abuse 24.0 5.45 3 141,717 Avg: 125,777 Control subjects for addicts (C) C-1 F 42 Breast cancer 15.0 N/A 1.0 84,546 C-2 M 32 MI, aneurism N/A N/A 8.0 89,652 C-3 M 61 Pneumonia, testicular tumor N/A N/A 8.0 65,833 C-4 F 44 Breast cancer N/A N/A 8.0 86,354 C-5 F 27 Mild hypoxia, arterial calcification, congenital heart defect, coronary artery atherosclerosis N/A N/A 1.5 78,656 C-6 F 33 SLE, sepsis N/A N/A 1.5 76,921 C-7 M 29 Leukemia, bacterial sepsis, hypoxia N/A N/A 1.5 89,732 Avg: 81,671 Control subjects for narcolepsy NBB-00-022 F 83 Acute MI 7.5 6.52 0.1 106,366 NBB-00-072 M 78 Kidney failure 18.0 5.84 0.1 88,404 NBB-00-142 F 82 MI 5.3 6.6 0.1 70,976 Avg: 88,582 Narcolepsy with cataplexy NBB-08023 F 66 Heart failure Amphetamine, 5.45 mg, 1 time/day Modafinil, 100 mg, 3 times/day 7.0 6.57 0.1 3276 3.6% control Sodium oxybate at 500 mg/ml, 2 times 6 ml/day Narcolepsy with cataplexy morphine-treated hypersomnia NBB-01064 F 85 Chronic pain syndrome with palliative sedation Morphine, 10 mg, 2 times/day (9 years) 3.4 6.77 0.1 16,834 19% control Modafinil, 200 mg, 1–2 times/day
Supplementary Materials
www.sciencetranslationalmedicine.org/cgi/content/full/10/447/eaao4953/DC1
Fig. S1. Western blots for Fig. 3 (D and E).
Table S1. Characteristics of animals used for morphine study.
Additional Files
- Supplementary Material for:
Opiates increase the number of hypocretin-producing cells in human and mouse brain and reverse cataplexy in a mouse model of narcolepsy
Thomas C. Thannickal, Joshi John, Ling Shan, Dick F. Swaab, Ming-Fung Wu, Lalini Ramanathan, Ronald McGregor, Keng-Tee Chew, Marcia Cornford, Akihiro Yamanaka, Ayumu Inutsuka, Rolf Fronczek, Gert Jan Lammers, Paul F. Worley, Jerome M. Siegel*
*Corresponding author. Email: jsiegel{at}ucla.edu
Published 27 June 2018, Sci. Transl. Med. 10, eaao4953 (2018)
DOI: 10.1126/scitranslmed.aao4953This PDF file includes:
- Fig. S1. Western blots for Fig. 3 (D and E).
- Table S1. Characteristics of animals used for morphine study.
