Opioid-induced decreases in rat brain adenosine levels are reversed by inhibiting adenosine deaminase

Abstract

Background: Opioids disrupt sleep and adenosine promotes sleep, but no studies have characterized the effects of opioids on adenosine levels in brain regions known to regulate states of arousal. Delivering opioids to the pontine reticular formation (PRF) and substantia innominata (SI) region of the basal forebrain disrupts sleep. In contrast, administering adenosine agonists to the PRF or SI increases sleep. These findings encouraged the current study testing the hypothesis that microdialysis delivery of opioids to the PRF or SI decreases adenosine levels in the PRF or SI, respectively.

Methods: A microdialysis probe was placed in the PRF of isoflurane anesthetized rats and perfused with Ringer's solution (control) followed by Ringer's solution containing morphine (0, 10, 30, 100, or 300 microm), fentanyl (100 microm), morphine (100 microm) and the adenosine deaminase inhibitor EHNA (100 microm), or naloxone (10 microm) and morphine (100 microm). Additional experiments measured adenosine levels in the SI before and during microdialysis delivery of morphine, fentanyl, and morphine plus EHNA.

Results: Morphine caused a significant (P < 0.05) concentration-dependent decrease in PRF adenosine levels. The significant decrease (-20%) in adenosine caused by 100 microm morphine was blocked by coadministration of naloxone. Fentanyl also significantly decreased (-13.3%) PRF adenosine. SI adenosine levels were decreased by morphine (-26.8%) and fentanyl (-27.4%). In both PRF and SI, coadministration of morphine and EHNA prevented the significant decrease in adenosine levels caused by morphine alone.

Conclusions: These data support the interpretation that decreased adenosine levels in sleep-regulating brain regions may be one of the mechanisms by which opioids disrupt sleep.

Fig. 1

Time course of stable adenosine measures. (A) Time course of pontine reticular formation (PRF) adenosine levels across 16 sequential brain dialysis samples collected during control dialysis with Ringer’s. Adenosine levels stabilized 120 min after dialysis probe placement in the PRF and remained stable for an additional 120 min. (B) There was no significant difference between average PRF adenosine levels during min 121–180 and min 181–240.

Fig. 2

Opioids decrease pontine reticular formation (PRF) adenosine levels. (A) Data summarize PRF adenosine levels during dialysis with Ringer’s and 10, 30,100 and 300 µM morphine. Asterisks indicate a significant (p<0.05) decrease from Ringer's control (0 µM). (B) The morphine (100 µM) induced decrease in PRF adenosine was blocked by coadministration of naloxone (10 µM). These data illustrate that adenosine levels during coadministration of morphine and naloxone were not statistically different from adenosine levels during dialysis with Ringer’s. (C) Dialysis with fentanyl (100 µM) caused a decrease in PRF adenosine levels. (D) A plate from a rat brain atlas illustrates a microdialysis probe placed in the PRF for drug delivery and collecting endogenous adenosine. The digitized rat brain stem section at right shows a representative microdialysis site (arrow) in the PRF.

Fig. 3

Morphine and fentanyl decreased adenosine levels in the substantia innominata (SI). (A) Microdialysis with morphine (100 µM) decreased SI adenosine levels. (B) Similarly, Student’s t-test revealed a significant decrease in SI adenosine levels during dialysis with fentanyl (100 µM). (C) A coronal brain plate (left) illustrates placement of a microdialysis probe in the SI. A cresyl violet stained brain section (right) confirms that the microdialysis site was localized to the SI.

Fig. 4

The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) prevented the morphine-induced decrease in adenosine. In both the pontine reticular formation (A) and the substantia innominata (B) coadministration of EHNA, which prevents the degradation of endogenous adenosine, overcame the decrease in adenosine caused by morphine alone.