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Background: ɣ-aminobutyric acid (GABA) facilitator valproic acid may be able to curb memory disruption induced by morphine exposure., Objective: The effects of the GABA facilitator valproic acid on the behavioral tolerance induced by morphine were investigated. Then hippocampal-dependent tasks named spatial-working and short-term memory procedures using the Y-maze apparatus were examined in morphine tolerant rats. Finally, the changes in the expression of hippocampal GABA-A receptors underlying morphine tolerance were also examined., Methods: Rats were treated with daily morphine injections, with or without distinct contextual pairing. To examine the effect of valproic acid on morphine tolerance expression, valproic acid was pretreated an hour before morphine. Spatial-working and short-term memory procedures using the Y-maze apparatus were examined in morphine tolerant rats. Afterwards the changes in the expression of hippocampal GABAα receptors using the quantitative real-time PCR and western blot techniques to detect GABArα subunits mRNAs and protein level were studied., Results: Our results showed that both learned and non-associative morphine tolerance influence short-term memory and the subjacent expression of GABArα mRNAs and protein level. Despite its attenuating effects on the development and expression of both learned and non-associative morphine tolerance, only associative morphine tolerance-induced memory dysfunction was ameliorated by valproic acid pretreatment. We also found that the expression of GABArα1, α2, α5 subunits mRNAs and GABAα protein level were affected heavier in associative morphine tolerant rats., Conclusion: Our data supports the hypothesis that unconditioned and learned morphine tolerance influences short-term memory and the expression of GABArα 1, α2, α5 mRNAs and GABArα protein level differently, and adds to our understanding of the behavioral and molecular aspects of the learned tolerance to morphine effects., Competing Interests: The authors have declared that no competing interests exist.

Studies have shown that following opiate withdrawal, the spontaneous discharge rate of locus coeruleus (LC) neurons remarkably increases. Combination of intrinsic mechanisms with extrinsic excitatory modulations mediates the withdrawal-induced hyperactivity of LC neurons. The nucleus paragigantocellularis (PGi) provides the main excitatory inputs to LC and plays a pivotal role in opiate withdrawal. In the present study the direct facilitatory role of PGi on opiate withdrawal-induced hyperactivity of LC neurons was investigated using a newly developed brain slice, containing both LC and PGi. HRP retrograde neuronal tracing was used to verify the existence of both LC and PGi neurons in the developed slice. The spontaneous discharge rate (SDR), resting membrane potential (RMP) and spontaneous excitatory post-synaptic currents (sEPSCs) were recorded in LC neurons using whole cell patch clamp recording. Results showed that the net SDR and the net RMP of LC neurons in slices containing both LC and PGi neurons are significantly higher than slices lacking intact (uncut) PGi inputs. Also, the frequency of sEPSCs in those LC neurons receiving PGi inputs significantly increased compared to the slices containing no intact PGi inputs. Altogether, our results propose that increase in PGi-mediated excitatory transmission might facilitate the opiate withdrawal-induced hyperactivity of LC neurons.

The effects of chronic morphine exposure on synaptic plasticity in the CA1 region of the hippocampal slice preparation using extracellular recordings of the population spike (PS) evoked in response to Schaffer collateral stimulation were studied. High frequency stimulation (HFS; 1X100 Hz) and theta pulse stimulation (TPS; 5 Hz trains for 3 min) were used as patterned activities. The results showed that in rats chronically treated with morphine (dependent group), TPS induced long-term depression (LTD) of PS in CA1 in the absence of in vitro morphine. This TPS-induced PS LTD was blocked in the presence of either AP5 (NMDAR antagonist) or CPX (A1 adenosine receptor antagonist) alone, but was not blocked when AP5 and CPX were co-applied. This TPS-induced PS LTD was also blocked in the presence of either 8-PT (a selective A1 adenosine receptor antagonist) or MRS1220 (a specific A3 receptor antagonist). Additionally, when TPS was applied prior to HFS, PS long-term potentiation (LTP) was blocked. However, when TPS was applied after HFS, there was no reversal of PS LTP in slices from dependent rats in contrast to controls which displayed reversal of LTP. Both the PS LTD and the absence of PS LTP reversal were blocked by in vitro application of morphine. It is concluded that morphine withdrawal was associated with greater depression of CA1 PS elicited by natural stimulus induced activity pattern. This effect was associated with changes in NMDA and adenosine receptors due to chronic morphine administration. Such an in vitro preparation could provide a novel paradigm to investigate withdrawal effects on synaptic plasticity.

Chronic morphine leads to dependence, tolerance, and neural apoptosis. Vitamin C inhibits the withdrawal syndrome in morphine-dependent subjects and prevents apoptosis in experimental models. Sodium-dependent vitamin C transporter (SVCT) type-2 is the main transporter for carrying vitamin C into the brain and neural cells. The mechanism(s) by which vitamin C inhibits morphine dependence in not understood. SVCT activity determines the vitamin C availably within the nervous system. We have examined the alterations in the expression of SVCT1, SVCT2, and its splice variants in morphine-tolerant rats. Morphine (20 mg/kg) was injected twice/day to male rats for either 7 or 14 days. The development of analgesic tolerance was assessed using tail-flick test. Lumbar spinal cord and the hippocampus were isolated for RNA extraction. Semiquantitative reverse transcriptase-polymerase chain reaction method was used to assess the levels of gene expression. Administration of morphine for 7 or 14 days reduced the expression level of SVCT2 in both hippocampus and dorsal lumbar spinal cord of rats. SVCT2 expression was reduced in vitamin C-, and vitamin C combined with morphine-treated animals. Results did not show SVCT2 splice variation. SVCT1 did not express in control or morphine-treated rats. It seems that reduced expression level of SVCT2 might be involved in the development of morphine side effects such as tolerance and dependency.

There are some reports indicating that adrenalectomy significantly potentiates morphine-induced analgesia. Since G-protein subunits have an important role in morphine effects at the cellular level and the exact mechanism(s) of adrenalectomy-induced morphine sensitization has not yet been clarified, the present study was designed to determine the changes in the levels of Galphai/o, Galphas, Gbeta mRNA involved in this phenomenon. All experiments were carried out on male Wistar rats. The tail-flick test was used to assess the nociceptive threshold and corticosterone levels were measured by radioimmunoassay as a marker of HPA function. The dorsal half of the lumbar spinal cord was assayed for the expression of G-protein subunits using semiquantitative PCR normalized to beta-actin gene expression. Results showed that morphine not only in 3 mg/kg, but also in a sub-effective dose (2 mg/kg) could affect the nociceptive threshold and induce an analgesic response in adrenalectomized (ADX) rats while 2 mg/kg morphine did not demonstrate analgesic properties in sham-operated animals. These effects were reversed with corticosterone replacement. Morphine increased plasma corticosterone concentration in a dose-dependent manner in sham-operated rats. Following adrenalectomy a significant increase in the mRNA levels of Galphai/o (79%) and Gbeta (96%) was observed in the dorsal portion of the lumbar spinal cord. In contrast, no significant changes were observed in the mRNA level of Galphas. In conclusion, our results demonstrate that the levels of the cellular components involved in morphine analgesia significantly increase in ADX animals. This may be at least partly responsible for adrenalectomy-induced morphine sensitization.

Opioids can induce analgesia and also hyperalgesia in humans and in animals. It has been shown that systemic administration of morphine induced a hyperalgesic response at an extremely low dose. However, the exact mechanism(s) underlying opioid-induced hyperalgesia has not yet been clarified. Here, we have investigated cellular events involved in low-dose morphine hyperalgesia in male Wistar rats. The data showed that morphine (0.01 microg i.t.) could elicit hyperalgesia as assessed by the tail-flick test. G(alphas) mRNA and protein levels increased significantly following exposure to the hyperalgesic dose of morphine. Furthermore, morphine at an analgesic dose (20 microg i.t.) significantly decreased cAMP levels in the dorsal half of the lumbar spinal cord, whereas the tissue cAMP levels were not affected by morphine treatment at a hyperalgesic dose. Intrathecal administration of nifedipine, an L-type calcium channel blocker, antagonized the hyperalgesia induced by the low-dose of morphine. Furthermore, pretreatment with the selective protein kinase C (PKC) inhibitor chelerytrine resulted in prevention of the morphine-induced hyperalgesia. KT 5720, a specific inhibitor of protein kinase A (PKA), did not show any effect on low-dose morphine-induced hyperalgesia. These results indicate a role for G(alphas), the PLC-PKC pathway, and L-type calcium channels in intrathecal morphine-induced hyperalgesia in rats. Activation of ordinary G(alphas) signaling through cAMP levels did not appear to play a major role in the induction of hyperalgesia by low-dose of morphine., ((c) 2007 Wiley-Liss, Inc.)

Ultra-low doses of morphine (UL-morphine) induce hyperalgesia, which is assumed to be mediated by stimulatory G proteins (G(alphas)) signaling pathway. G(alphas) pathway inhibition and chronic stress both attenuate development of tolerance to analgesic effect of morphine. This study evaluated the effect of chronic stress on UL-morphine-induced hyperalgesia to find out if chronic stress interacts with the G(alphas) signaling pathway. Repeated daily forced swim stress was applied to induce chronic stress. UL-morphine (1 microg/kg, intraperitoneal)-induced hyperalgesia was assessed using the tail-flick test on day 6, in male rats that during days 1-5 received different treatments of swim stress, dexamethasone, swim stress following adrenalectomy (ADX) or swim stress after sham operation. Chronic stress by itself induced hyperalgesia in control and sham-operated rats but inhibited UL-morphine-induced hyperalgesia. In ADX animals, chronic stress did not produce hyperalgesia and could not inhibit UL-morphine-induced hyperalgesia. Chronic dexamethasone produced hyperalgesia but did not change the UL-morphine-induced hyperalgesia. Inhibition of UL-morphine hyperalgesia by chronic stress suggests that chronic stress interacts with the G(alphas) signaling pathway, which is responsible for UL-morphine-induced hyperalgesia. The absence of this effect in the ADX-rats or after repetitive dexamethasone administration demonstrates that hypothalamic-pituitary-adrenal (HPA) axis activation is necessary for controlling UL-morphine-induced hyperalgesia. Finally, the interaction of stress with the G(alphas) signaling pathway may provide an explanation for the inhibitory effect of stress on development of tolerance to the analgesic effect of morphine.

It has been shown that systemic administration of morphine induced a hyperalgesic response at an extremely low dose. We have examined the effect of nifedipine, as a calcium channel blocker, on morphine-induced hyperalgesia in intact and adrenalectomized rats and on hypothalamic-pituitary-adrenal axis activity induced by ultra-low dose of morphine. To determine the effect of nifedipine on hyperalgesic effect of morphine, nifedipine (2 mg/kg i.p. and 10 microg i.t.) that had no nociceptive effect, was injected concomitant with morphine (1 microg/kg i.p. and 0.01 microg i.t. respectively). The tail-flick test was used to assess the nociceptive threshold, before and 30, 60, 120, 180, 240 and 300 min after drug administration. The data showed that low dose morphine systemic administration could produce hyperalgesic effect in adrenalectomized rats equivalent to sham-operated animals while intrathecal injection of morphine only elicited hyperalgesia in sham-operated animals. Nifedipine could block morphine-induced hyperalgesia in sham and adrenalectomized rats and even a mild analgesic effect was observed in the adrenalectomized group which was reversed by corticosterone replacement. Systemic administration of low dose morphine produced significant increase in plasma level of corticosterone. Nifedipine has an inhibitory effect on morphine-induced corticosterone secretion. Thus, the data indicate that dihydropyridine calcium channels are involved in ultra-low dose morphine-induced hyperalgesia and that both the pattern of morphine hyperalgesia and the blockage of it by nifedipine are modulated by manipulation of the hypothalamic pituitary adrenal axis.

It is previously reported that the HPA axis plays role in the inhibitory effect of pain on tolerance development to analgesic effect of opioids. The present study was designed to investigate whether the chronic co-administration of dexamethasone as a glucocorticoid is also able to prevent or reverse analgesic tolerance to morphine and to compare the expression of G(alphai/o) and G(beta) subunits of G proteins in the context of chronic dexamethasone, development of morphine tolerance and their combination. Analgesic tolerance to morphine was induced by chronic intraperitoneally (i.p.) administration of morphine 20 mg/kg to male Wistar rats weighing 200-240 g within 4 consecutive days and analgesia was assessed using tail-flick test. Chronic dexamethasone was applied using 4 daily i.p. injections. Lumbar spinal tissues were assayed for the expression of G(alphai/o) and G(beta) proteins using "semiquantitative PCR" normalized to beta-actin gene expression. Results showed that chronic administration of dexamethasone could reduce and reverse the development of tolerance in rats that received chronic i.p. injections of morphine. Chronic administration of dexamethasone significantly increased the expression of G(alphai/o), while chronic administration of morphine did not change its expression. The expression of G(beta), however, was increased after the chronic administration of morphine, but did not change after the administration of chronic dexamethasone. None of these increases were observed when morphine and dexamethasone were co-administered. We conclude that the development of tolerance to analgesic effect of morphine could be prevented and reversed by dexamethasone co-administration. The increase in G(alphai/o) genes expression produced by chronic dexamethasone may facilitate the opioid signaling pathway and compensate for morphine-induced tolerance.