C57BL/6J mice become morphine-dependent after 4 days of administration of increasing doses of morphine; the morphine-dependent mice develop signs of withdrawal within 18 hours of their last morphine dose, and naloxone administration rapidly induces substantial withdrawal symptoms (Chu et al., 2009
). To characterize opiate-induced metabolomic changes, brainstem tissue was prepared from C57BL/6J mice placed into 4 treatment groups (n=8 per group) (): 1) Dependence: tissue was harvested 1 hour after the last morphine dose on day 4; 2) Naloxone-induced withdrawal: the opiate receptor antagonist naloxone (10 mg/kg s.c.) was administered to morphine-dependent mice 1 hour after their last morphine dose on day 4, and then tissue was harvested 10 minutes later; 3) Natural withdrawal: tissue was harvested 18 hours after their last morphine dose on day 4, which is when withdrawal symptoms are maximal; 4) Control: saline injections were administered over the 4 day period. Within the 32-brainstem tissues analyzed, 1300 metabolite peaks were identified. When the dependence, naloxone-induced withdrawal, or natural withdrawal samples were compared to the control samples, adenosine was the only metabolite whose abundance was significantly altered in all 3 conditions after morphine administration (). The identity of adenosine as the differentially reduced metabolite in all 3 conditions was confirmed using a chemical standard and by LC/MS/MS analysis (). Brainstem adenosine abundance was significantly lower (2-fold, p<0.00005) in all 3 conditions associated with morphine administration relative to that of control mice. Thus, opiate exposure induces a 50% decrease in brainstem adenosine abundance. This represents a very significant metabolomic response, since adenosine is an important neuromodulator whose intracellular and extra-cellular concentration is very tightly controlled by transporters and by enzymes regulating adenosine metabolism [reviewed in (Boison, 2006
)]. Adenosine abundance was significantly decreased during the period of morphine dependence, and this decrease is maintained during the 18-hour period of narcotic drug abstinence when these mice manifest maximal signs of withdrawal.
Figure 1 A) The 4-day protocol for developing morphine dependence, and for inducing natural or naloxone-induced withdrawal is shown. B) Adenosine abundance (+ SEM) in brainstem tissue obtained from 4 groups of C57BL/6J mice (n=8 per group). C) Adenosine abundance (more ...)
Figure 2 Confirmation of the identity of adenosine in extracts using a chemical standard. A) The extracted ion chromatogram of adenosine in a brainstem extract (red) overlapped with that of an adenosine standard (black), which indicates that they have identical (more ...)
Since 129Sv1 mice do not develop physical dependence after 4 days of morphine administration (Chu et al., 2009
), metabolomic changes in brainstem tissues in these mice were also examined during the dependence and naloxone-induced withdrawal states. In contrast to C57BL/6J mice, no metabolite exhibited a significant change in 129Sv1 brainstem tissue. In particular, adenosine abundance was un-altered () in 129Sv1 mice; while adenosine abundance was decreased in the brainstem of C57BL/6J mice during the dependent state, which were subject to the same morphine administration protocol and at the same time as the 129Sv1 mice. We previously demonstrated that co-administration of ondansetron (1 mg/kg IP with morphine prevented morphine tolerance and dependence in C57BL/6J mice (Chu et al., 2009
). However, ondansetron co-administration with morphine did not alter the decrease in brainstem adenosine abundance that is induced by morphine and naloxone treatment (). Thus, there is a specific decrease in brainstem adenosine levels in C57BL/6J mice during the morphine dependence and withdrawal states; this metabolomic change specifically occurs in the opiate-dependent strain. While ondansetron reduces behavioral aspects of dependence, it acts distal to the site where opiates alter adenosine abundance.
Adenosine deaminase and adenosine kinase (ADK) can reduce adenosine by forming inosine and AMP, respectively. However, ADK is the key regulator of adenosine metabolism in the adult brain (Boison, 2006
). Consistent with ADK playing an important role in the opiate response, inosine abundance in brainstem tissues during the periods of morphine dependence or during the two different withdrawal states was not altered (). To study the role of adenosine and ADK in opiate responses, we characterized opiate responses in two mouse strains with altered levels of ADK expression (Li et al., 2008
). In both of these lines, the endogenous ADK, which is subject to dynamic expression changes (Masino et al., 2011
) (Studer et al., 2006
), has been replaced by a constitutively over-expressed transgene (Li et al., 2007
) (Li et al., 2008
). Both lines were maintained on the C57BL/6J background that exhibits a high degree of opiate dependence. Adk-tg
mice have globally increased ADK expression in brain, including brainstem; while fb-Adk-def
have an identical level of ADK over-expression of ADK throughout the entire basal and midbrain regions, but have reduced ADK expression within the entire dorsal telencephalon (Li et al., 2008
) (). We first measured the jumping behavior precipitated by naloxone administration to opiate dependent control, Adk-tg
, and fb-Adk-def
mice. There was a very significant reduction (P<0.0004) in the withdrawal response exhibited by Adk-tg
mice relative to control C57BL/6J mice (). The similarly reduced withdrawal response in transgenic mice with increased (Adk-tg
) or decreased (fb-Adk-def)
forebrain ADK expression (p>0.05) indicates that other brain regions affect the withdrawal response.
Figure 3 Inosine abundance (± SEM) in C57BL/6J brainstem tissue obtained from the indicated 4 groups of mice (n=8 per group). The p-value and fold change, which is calculated relative to that in the control mice for each of the 3 groups analyzed, is also (more ...)
Figure 4 A) Representative immunohistochemical images of sagittal brain sections from wildtype (C57BL/6J), fb-Adk-def and Adk-tg mice. The sections were stained with diamino-benzidine hydrochloride (DAB) for ADK immunoreactivity. B) C57BL/6J, Adk-tg, and fb-adk-def (more ...)
Then, adenosine abundance in the brainstem of control C57BL/6J and fb-Adk-def mice was measured in the basal state and in morphine dependent mice after naloxone-induced withdrawal. As before, adenosine abundance was decreased in C57BL/6J mice during naloxone-precipitated withdrawal (p=0.028) relative to basal. Although adenosine abundance in brainstem was reduced in fb-Adk-def mice in the basal state relative to control C57BL/6J mice, it was not further decreased in the withdrawal state, which is consistent with the elimination of the endogenous Adk-gene (). This indicates that ADK activity affects the development of narcotic drug dependence and the level of adenosine in the brainstem. It is also noteworthy that the basal adenosine levels in both 129Sv1 and fb-Adk-def mice were lower than those in C57BL/6J mice, which exhibited a greater level of narcotic dependence after four days of opiate administration ( and ). Furthermore, unlike C57BL/6 mice, the brainstem adenosine levels in 129Sv1 and fb-Adk-def mice were not changed after morphine administration.
It was surprising that the opiate-induced change in the brainstem adenosine level was not altered by ondansetron, especially since ondansetron alleviated opiate dependence and withdrawal symptoms. However, it was possible that ondansetron could act downstream of the opiate-induced change in adenosine, possibly through an effect on adenosine receptors. To investigate this possibility, we examined the effect that drugs acting on adenosine receptors had on opiate-induced behaviors. Consistent with the results observed by others (Zarrindast et al., 1999
), administration of an adenosine receptor1
) agonist significantly attenuated behavioral evidence of naloxone-induced opioid withdrawal in our protocol. Somewhat surprisingly, administration of an adenosine A2a
) antagonist also decreased opioid withdrawal symptoms (). Moreover, both agents significantly ameliorated morphine induced hyperalgesia, another opioid withdrawal complication (). These results demonstrate that adenosine receptors have a strong effect on reducing withdrawal behaviors.