Microinjection of the vehicle dimethyl sulfoxide (DMSO) into the periaqueductal gray modulates morphine antinociception.

Dimethyl sulfoxide (DMSO) is commonly used as a solvent for water-insoluble drugs. Given that DMSO has varying cellular and behavioral effects ranging from increased membrane permeability to toxicity, microinjection of DMSO as a vehicle could confound the effects of other drugs. For example, DMSO is often used as a vehicle for studies examining the neurochemical mechanisms underlying morphine antinociception. Given that the ventrolateral periaqueductal gray (vlPAG) plays a major role in morphine antinociception and tolerance, the effects of DMSO on morphine antinociception mediated by the vlPAG needs to be evaluated. The present experiment tested whether co-administration of DMSO (0, 0.2, 2, or 20%) would alter the antinociceptive effect of microinjecting morphine into the vlPAG. DMSO had no effect on nociception when microinjected into the vlPAG alone, but 2% DMSO enhanced morphine potency when co-administered with morphine. In contrast, twice daily microinjections of DMSO (5 or 20%) for two days reduced the potency of subsequent microinjections of morphine into the vlPAG--an effect that persisted for at least one week. A similar rightward shift in the morphine dose-response curve was caused by morphine tolerance. Co-administration of morphine and DMSO during the pretreatment did not cause a greater shift in the morphine dose-response curve compared to morphine pretreated alone. In conclusion, DMSO can alter morphine antinociception following both acute (enhancement) and chronic (inhibition) administration depending on the concentration. These data reinforce the need to be cautious when using DMSO as a vehicle for drug administration.

Behavioral and electrophysiological evidence for opioid tolerance in adolescent rats.

Morphine and other opiates are successful treatments for pain, but their usefulness is limited by the development of tolerance. Given that recent studies have observed differential sensitivity to drugs of abuse in adolescents, the aim of this study was to assess antinociceptive tolerance to morphine in adolescent rats using both behavioral and cellular measures. Early (28-35 days postnatal) and late (50-59 days) adolescent and adult (73-75 days) male rats were injected with morphine (5 mg/kg, s.c.) or saline twice a day for two consecutive days. On Day 3, tolerance to morphine was evident in morphine-pretreated rats when tested on the hot plate test. Although baseline latencies for the early compared to late adolescent rats were faster, the magnitude of the shift in ED(50) for morphine was similar for the two adolescent groups. However, the shift in ED(50) tended to be greater in adolescent compared to adult rats. Subsequent to behavioral testing, whole cell patch-clamp recordings were made from ventrolateral PAG neurons. The opioid agonist, met-enkephalin (ME), activated similar outward currents in PAG neurons of early and late adolescent rats. However, reversal potentials of ME-induced currents were shifted to more hyperpolarized potentials in cells from morphine-pretreated rats. In addition, ME induced larger currents in morphine-pretreated rats with faster hot plate latencies compared to the mean (more tolerant) than in rats with slower latencies. These results indicate that repeated intermittent administration of morphine produces tolerance in adolescent rats that is associated with novel changes in opioid-sensitive ventrolateral PAG neurons.

Analgesic tolerance to microinjection of the micro-opioid agonist DAMGO into the ventrolateral periaqueductal gray.

Repeated administration of the relatively low-efficacy micro-opioid receptor agonist morphine induces tolerance to its antinociceptive effects. High-efficacy agonists such as D-Ala2NMePhe4,Gly-ol5 (DAMGO) have been shown to be less effective at producing tolerance, suggesting that different neural mechanisms underlie tolerance to these agonists. However, the correlation between agonist efficacy and tolerance development has not been examined within the ventrolateral periaqueductal gray (vPAG), a brain area known to be crucial for the development of morphine tolerance. The current studies examined whether tolerance to DAMGO occurs within the vPAG, and whether repeated treatment with DAMGO into the vPAG alters the development of morphine tolerance. The results showed that repeated vPAG microinjections of DAMGO induced robust tolerance and cross-tolerance to morphine. Further, co-administration of a low dose of DAMGO with morphine potentiated morphine tolerance. These findings indicate that similar mechanisms underlie tolerance to morphine and DAMGO within the vPAG.

Morphine antinociceptive potency on chemical, mechanical, and thermal nociceptive tests in the rat.

UNLABELLED: Many tests are used to assess nociception in laboratory animals. The objective of this study was to compare morphine potency across tests. Rats were injected with saline or morphine (1-20 mg/kg SC), and nociception was assessed 15-20 min later. A consistent definition of antinociception-a change in response greater than 4 times the standard deviation above the mean for the saline-treated controls-was used to compare morphine potency on different tests. These data revealed 4 things. 1) Morphine potency was greatest on the paw pressure, hot plate, and tail withdrawal tests and lowest on the formalin test. 2) Stimulus intensity had no effect on morphine potency on the hot plate (ED50 = 4.5, 2.8, and 2.6 mg/kg for 49 degrees C, 52 degrees C, and 55 degrees C tests, respectively) or tail withdrawal tests (ED50 = 2.9 and 2.6 for 48 degrees C and 52 degrees C water, respectively). 3) Assessment of morphine potency using a within-subjects cumulative dosing procedure resulted in lower ED50 values compared to data collected using a between-subjects design (hot plate: 2.6 vs 4.9; tail withdrawal: 2.6-2.9 vs 5.7 mg/kg). 4) Adjusting the cutoff value from 4 to 5, 6, 7, and 8 standard deviations greater than the mean resulted in a progressive increase in ED50 values. These data demonstrate that morphine potency is dependent, in part, on the nociceptive test even when all other factors (eg, species, strain, age, gender, and cutoff value) are held constant. PERSPECTIVE: The ability of morphine to block nociception is influenced by many factors. The present study shows that the test used to assess nociception, but not the stimulus intensity, can have a dramatic effect on morphine potency. This finding shows that morphine potency varies depending on how pain is assessed.

Antinociceptive tolerance revealed by cumulative intracranial microinjections of morphine into the periaqueductal gray in the rat.

The periaqueductal gray (PAG) appears to play a key role in morphine antinociception and tolerance. The objective of this manuscript is to develop a cumulative dose microinjection procedure so the hypothesized role of the PAG in morphine antinociceptive tolerance can be assessed using dose-response analysis. Rats were implanted with a guide cannula into the ventrolateral PAG. Microinjection of cumulative half log doses of morphine (0.32, 1, 3.2, and 10 micro g/0.4 micro l) produced antinociception on the hot plate test only at the two highest doses. Microinjection of quarter log doses of morphine into the PAG (1, 1.8, 3.2, 5.6, and 10 micro g/0.4 micro l) resulted in an ED(50) for antinociception of 1.8 mug. Systemic administration of the opioid antagonist naloxone increased the morphine ED(50) to 9.0 micro g. Repeated microinjections of saline into the PAG had no effect on nociception. Pretreatment with twice daily injections of morphine, either systemically (5 mg/kg, s.c.) or into the PAG (5 micro g/0.4 micro l), for 2 days produced a two-fold increase in the ED(50) for morphine antinociception. These data validate the use of an intracranial cumulative dose procedure to assess morphine potency and demonstrate that microinjection of morphine into the PAG is sufficient to produce tolerance.

Tolerance to repeated morphine administration is associated with increased potency of opioid agonists.

Tolerance to the pain-relieving effects of opiates limits their clinical use. Although morphine tolerance is associated with desensitization of mu-opioid receptors, the underlying cellular mechanisms are not understood. One problem with the desensitization hypothesis is that acute morphine does not readily desensitize mu-opioid receptors in many cell types. Given that neurons in the periaqueductal gray (PAG) contribute to morphine antinociception and tolerance, an understanding of desensitization in PAG neurons is particularly relevant. Opioid activity in the PAG can be monitored with activation of G-protein-mediated inwardly rectifying potassium (GIRK) currents. The present data show that opioids have a biphasic effect on GIRK currents in morphine tolerant rats. Opioid activation of GIRK currents is initially potentiated in morphine (EC(50)=281 nM) compared to saline (EC(50)=8.8 microM) pretreated rats as indicated by a leftward shift in the concentration-response curve for met-enkephalin (ME)-induced currents. These currents were inhibited by superfusion of the mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA) suggesting that repeated morphine administration enhances agonist stimulation of mu-opioid receptor coupling to G-proteins. Although supersensitivity of mu-opioid receptors in the PAG is counterintuitive to the development of tolerance, peak GIRK currents from tolerant rats desensitized more than currents from saline pretreated rats (56% of peak current after 10 min compared to 15%, respectively). These data indicate that antinociceptive tolerance may be triggered by enhanced agonist potency resulting in increased desensitization of mu-opioid receptors.