Seasonal variation of morphine physical dependence

The characteristics of morphine physical dependence in ground squirrels ($\text{Citellus lateralis}$) were examined during each of the four annual seasons. The results showed that this rodent hibernator exhibits a strong and characteristic naloxone-precipitated abstinence syndrome during its non-hibernation state, irrespective of the season. Although qualitatively unchanged throughout the year, the abstinence syndrome showed clear quantitative seasonal differences. These differences were evident in terms of both the number of occurences of particular signs and the percentage of the morphine-dependent population exhibiting them.     

Differential effect of environmental temperature on morphine physical dependence and abstinence

1) Ambient temperature (T_a) significantly influenced the display of 4 of the 14 naloxone-precipitated withdrawal signs (nesting, flat posture, vocalization, dyspnea) in morphine-dependent, non-hibernating ground squirrels ($\text{Citellus}$$\text{lateralis}$).2) Analysis of variance performed on the six quantified signs revealed that T_a during withdrawal, but not during the development of physical dependence, was a significant factor in determining the expression of two signs (nesting and vocalization).3) The interaction between the influence of T_a during the periods of morphine administration and abstinence was a significant factor in determining the expression of nesting behavior, a finding that is consistent with the natural role of nesting as a behavioral thermoregulatory response.4) We conclude that environmental temperature modulates the expression of selected components of the naloxone-precipitated abstinence syndrome in $\text{C. lateralis}$ without exerting a measurable influence on the development of morphine physical dependence itself.     

Inability of cyclo (Leu-Gly) to facilitate the development of tolerance to and physical dependence on morphine in the rat

The effect of cyclo (Leu-Gly), an analog of melanotropin release inhibition factor on the development of tolerance to and physical dependence on morphine in the rat was investigated. Administration of cyclo (Leu-Gly) (1 μg/rat/day) prior to and during morphine pellet implantation failed to facilitate the development of tolerance to the analgesic and hypothermic effects of morphine. Similarly the development of dependence on morphine was not facilitated by cyclo (Leu-Gly) as evidenced by changes in body weight and body temperature observed during abrupt withdrawal of morphine. These studies do not lend support to the previous observations that cyclo (Leu-Gly) and other related peptides facilitate the development of tolerance to and physical dependence on morphine.     

Possible role of cyclic AMP and dopamine in morphine tolerance and physical dependence.

In chronically morphinized rats undergoing naloxone induced withdrawal the cerebellar Cyclic 3′, 5′ adenosine monophosphate (Cyclic AMP) was significantly higher than the controls. The cerebellar dopamine (DA) and norepinephrine (NE) were decreased, elevated or unchanged depending on the duration of morphine treatment. The corpus striatal DA levels during withdrawal were markedly elevated and the striatal cyclic AMP levels were unchanged. The NE levels in the striatal tissue were either elevated or unchanged depending upon the duration of morphine administration. In sharp contrast to the chronically morphinized rats undergoing naloxone induced withdrawal, the rats made morphine dependent over a period of eight weeks showed quite moderate changes in the striatal and cerebellar cyclic AMP and DA levels. Thus alterations in the DA and the cyclic AMP levels in the central nervous system (CNS) may play an important role in the naloxone induced stereotyped morphine withdrawal behavior.     

The opioid peptide analogue biphalin induces less physical dependence than morphine

We compared the physical dependence liability of biphalin, a dimeric enkephalin analogue that possesses high antinociceptive activity, with that of morphine in equipotent intravenous doses. Naloxone challenge produced severe withdrawal signs after a 5-day infusion of morphine but only minor withdrawal signs after a 5-day biphalin infusion. In a cross-dependence study, biphalin did not suppress body weight loss after morphine withdrawal, but successfully suppressed weight loss after pentazocine withdrawal. These data support consideration of biphalin as a new analgesic with a novel pharmacological profile and minimum dependence liability.     

The effect of mepyramine on the development of morphine tolerance and physical dependence in mice.

Latency relaxation (LR) as well as resting tension and twitch tension of frog toe muscles are studied in an isotonic solution (= 1 T) and in solutions made hypotonic by leaving out the appropriate amounts of NaCl and KCl (0.54 T and 0.76 T). In hypotonic solutions there is an increase in peak twitch tension as well as a decrease in the depth of the LR: the resting tension is increased at sarcomere lengths which are greater than 2.8 μm and is decreased at sarcomere lengths which are less than this value. The behaviour of twitch tension is discussed with respect to the influence of the sarcoplasmic ionic strength on the interaction between the contractile filaments. Concerning the decrease in both the LR and the resting tension, it is assumed that these effects are induced osmotically, the tension of the membranes of the longitudinal sarcoplasmic reticulum being the particular parameter which is influenced.     

Effect of cyclic nucleotides and phosphodiesterase inhibition on morphine tolerance and physical dependence.

The effects of cyclic nucleotides and theophylline were assessed in mice rendered tolerant to and physically dependent on morphine by the pellet implantation procedure. Tolerance was quantified by the increase in amount of morphine to produce analgesia and dependence by the decrease in amount of naloxone to precipitate withdrawal jumping. By these criteria, pretreatment with a single intravenous injection of cyclic 3′, 5′-adenosine monophosphate (cAMP) was found to enhance markedly tolerance and dependence development. Repeated injections of theophylline were also affective. Cycloheximide and beta-adrenergic blockers prevented the accelerating effect of cAMP and with more frequent administration also decreased the development of tolerance and dependence. It is concluded that cAMP may have a role in morphine tolerance and dependence development.     

Morphinome--a meta-analysis applied to proteomics studies in morphine dependence

This review is a meta-analysis of data describing proteins regulated by morphine influence studied worldwide across last years administration. Up to date (July 2010), 15 studies concerning this subject have been published. Animal models, examined brain structures, the route of morphine administration and proteomic platforms used for identification of differentially expressed proteins were described. Standardization of obtained results allowed for creation of database of proteins, whose expression was altered by morphine administration (www.addiction-proteomics.org). Their analysis by tools available in Celera Panther Database was possible too. Proteins, which seem to be the most promising candidates for further research, due to their consistent appearance in different studies, were indicated. Created database may facilitate further studies by providing a possibility to compare results obtained during different experiments. At the end, dynamic picture of proteome after morphine administration, which emerges from the obtained results, is discussed and need for standardization of proteomics experiments is stressed. As meta-analysis is a very powerful tool for evaluation and comparison of multiple data. We believe this approach will be useful in the nearest future to extract vital information from a vast number of similar publications. Morphinome database created already by our group is a comfortable tool for validation and verification of new data received after morphine influence on proteomes investigations. It gives a chance for fast comparison of results without hours spent on life science literature mining.     

New approaches to study the development of morphine tolerance and dependence

Morphine is now believed not to cause tolerance and dependence when it is appropriately used in clinic. However, in terminal cancer pain, patients' analgesic tolerance to morphine is developed due to the use of high doses of morphine for complete blockade of pain. At higher doses, morphine has more opportunity to show serious side effects, which worsens quality of life (QOL), and leads to the use of potent analgesic adjuvants to reduce the morphine dosage. Here we attempt to summarize recent studies of the molecular basis of morphine tolerance and dependence, and to discuss whether these mechanisms could provide new molecular targets as analgesic adjuvants. They include protein kinase C inhibitor, opioid agonist with low RAVE value, and antagonists of antiopioid receptors (GluRepsilon1 or nociceptin/OFQ receptor). In addition, we demonstrate new approaches to find further candidates of such molecular targets. These approaches include the visualization of neuronal networks in the downstream of opioid neurons by use of the WGA transgene technique and the single cell dissection technique to get new genes involved in plasticity during morphine tolerance and dependence.     

Pharmacological manipulation of gamma-aminobutyric acid (GABA) in morphine analgesia,tolerance and physical dependence

The findings from our laboratory indicated that pharmacological manipulations of GABA system modified morphine analgesia, tolerance and physical dependence. Elevating brain levels of GABA by slowing its destruction with aminooxyacetic acid not only antagonized the analgesic action of morphine in both non-tolerant and tolerant mice, but also enhanced the development of tolerance and physical dependence. On the other hand, blockade of postsynaptic sites of GABA receptors by bicuculline resulted in an inhibition of tolerance and dependence development. Administration of 2,4-diaminobutyric acid, an inhibitor of GABA uptake in the neurons, antagonized morphine analgesia in both non-tolerant and tolerant mice. However, it did not modify naloxone precipitated withdrawal jumping. On the contrary, β-alanine, an inhibitor of the GABA uptake process in glial cells, potentiated naloxone precipitated withdrawal jumping in morphine dependent mice, but it had no effect on morphine antinociception in both non-tolerant and tolerant mice.     

Tolerance to morphine in the rat: its prevention by naloxone.

Development of tolerance after a single injection of morphine in the Wistar-Lewis rat can be estimated by the attenuation of the response to a second injection of morphine given three days later. If naloxone is given 35 minutes after the first morphine injection and after the appearance of measurable analgesia, attenuation of the effects of the second morphine injection is not seen. It appears that naloxone blocks the development of tolerance to morphine even if given after the morphine-receptor interaction responsible for analgesia has been initiated. The temporal relationship between the prior injection of morphine and the subsequent administration of naloxone is being explored.     

Tolerance to morphine induced by chronic mild environmental stressors

Pain-sensitivity as well as the analgesic and thermoregulatory effects of morphine were studied after two different types of chronic environmental stresses in rats (extra stimulation of newborns for 21 days, or social isolation for a month in adult age). The basal pain sensitivity and the base-line body temperature were similarly affected after the two interventions: an increased tail-flick latency, a decreased hot-plate latency and a decreased body temperature were noted. The analgesic and thermoregulatory effects of morphine were uniformly reduced in rats exposed to either stress. These findings suggest a common effect of various non painful mild environmental stresses on the activity of the endogenous opioid system.     

Tolerance to effects of clonidine and morphine on sulfobromophthalein disposition in mice

Chronic treatment of mice with clonidine or morphine caused tolerance to the analgesic and thermoregulatory effects of these drugs. After chronic morphine, mice also became tolerant to the analgesic and thermoregulatory effects of clonidine. Cross tolerance to the hypothermic effect of morphine was demonstrated after chronic clonidine administration, but no diminution of morphine-induced analgesia could be shown. Morphine and clonidine acutely increased the retention of sulfobromophthalein (BSP) in plasma and liver. Chronic dosing with morphine or clonidine caused partial tolerance and cross-tolerance to the rise in hepatic BSP caused by an acute challenge with either agonist. However, both drugs elevated plasma BSP levels similarly in tolerant and non-tolerant mice. Thus, regimens which readily induced tolerance to the analgesic and hypothermic effects of morphine or clonidine were only partially effective in modifying the acute hepatobiliary effects of these drugs.     

Rat brain proteome in morphine dependence

The aim of this study was to reveal potential markers associated with drug dependence, using the proteomic approach. Gels containing samples derived from morphine-treated and control animals were compared and analyzed. Inspection of protein profiles, following TCA/acetone precipitation and the use of nano-scale liquid chromatography coupled to tandem mass spectrometry, allowed for identification of eleven potential dependence markers, mainly cytoplasmic and mitochondrial enzymes, e.g. proteins that belong to GTPase and GST superfamilies, ATPase, asparaginase or proteasome subunit p27 families.     

Effect of naltrindole on the development of physical dependence on morphine in mice: A behavioral and biochemical study

The effect of pretreatment with a δ opioid receptor antagonist, naltrindole (NTI), on the development of physical dependence on morphine was investigated in mice. Several withdrawal signs, an increase in cortical noradrenaline (NA) turnover and a decrease in dopamine (DA) turnover in the limbic forebrain were observed following naloxone challenge in morphine-dependent mice. Pretreatment with NTI (0.3–5 mg/Kg, S.c.) during chronic morphine treatment dose-dependently suppressed the behavioral and biochemical changes after withdrawal. The blocking effects of NTI suggest that δ opioid receptors may play a significant role in modulating the development of physical dependence on morphine.     

Increased CNS sensitivity to flurothyl as a measure of physical dependence in mice following morphine,phenobarbital, and ethanol treatment

Male C57BL/6 mice were administered either morphine, phenobarbital or ethanol in their drinking water in order to make them dependent. During withdrawal onset of myoclonus, clonus and tonus was evaluated with flurothyl, a convulsant inhalant. Morphine, phenobarbital and ethanol treated subjects displayed significantly lower latencies for myoclonic and clonic convulsive behavior when compared with their respective controls. The increased CNS excitability observed is characteristic of physical dependence and the flurothyl technique employed is discussed in light of its broad applicability to a number of agents characterized by their dependence producing properties.     

The role of T-type calcium channels in morphine analgesia,development of antinociceptive tolerance and dependence to morphine,and morphine abstinence syndrome

Involvement of T-type voltage dependent Ca2+ channels (VDCCs) on morphine antinociception, in the development of tolerance and dependence to morphine, and naloxone-precipitated abstinence syndrome in morphine dependent mice was examined by using mibefradil, a T-type VDCCs blocker. Mice were rendered tolerant and dependent on morphine by subcutaneous (s.c.) implantation of a morphine pellet containing 75 mg of morphine base for 72 hr. The tail-flick test was used to assess the nociceptive threshold. Coadministration of acute mibefradil (10 mg/kg, i.p.) with morphine enhanced the antinociceptive effects of acute morphine. Repeated mibefradil administration (10 mg/kg, i.p., just before, 24 and 48 hr after morphine pellet implantation) completely blocked the development of tolerance to the antinociceptive effect of morphine and even by this effect reached supersensitivity to morphine. However, repeated mibefradil treatment did not alter the development of dependence to morphine assessed by the A(50) values of naloxone (s.c.) required to precipitate withdrawal jumping 72 hr after morphine pellet. But, acute mibefradil (10, 30, and 50 mg/kg, i.p.) dose dependently decreased the expression of morphine abstinence syndrome when given directly 30 min prior to naloxone (0,05 mg/kg, s.c.) 72 hr after morphine pellet. These results indicate a critical role of T-type VDCCs in morphine antinociception, the development of tolerance to the antinociceptive effects of morphine and in morphine abstinence syndrome.     

Tolerance to morphine analgesia and immobility measured in rats by changes in log-dose-response curves

Using the bar and tailflick tests, morphine log-dose-response (LDR) curves were determined for immobility and analgesia, respectively, in male Wistar rats following 15 daily i.p. injections of 0, 20, or 200 mg/kg of morphine sulfate. The LDR curves for the two measures were qualitatively similar. Chronic morphine treatment resulted in a shift to the right and flattening of both curves. These results indicate that the flattening of the LDR curve in morphine tolerant rats is general to a number of opiate effects, and raise the possibility that both morphine-produced immobility and analgesia are subserved, in part, by a similar mechanism. In addition, after a test dose of 900 mg/kg, more rats in the 20 mg/kg than in the 200 mg/kg treatment group died of convulsions. Thus, tolerance developed to lethality produced by the convulsive effects of opiates.     

The role of mu1 receptor in physical dependence on morphine using the mu receptor deficient CXBK mouse.

It is known that the CXBK inbred strain of mouse is deficient in mu1 opioid receptors, whereas the strain has a delta opioid receptor population that is less consistently altered. In the present study, we compared physical dependence on morphine between CXBK and C57BL/6 mice. Both strains of mice were treated with morphine-admixed food for 5 days. During the treatment, the two strains of mice showed no signs of toxicity. There was no significant difference in morphine intake during the treatment between CXBK and C57BL/6 mice. After the treatment, the withdrawal was precipitated by injecting naloxone (0.01-30 mg/kg, s.c.). CXBK mice showed weight loss, diarrhea and ptosis, but not jumping and body shakes after low dose of naloxone. Whereas, C57BL/6 mice showed weight loss, diarrhea, ptosis, body shakes and jumping. These results suggest that naloxone-precipitated weight loss, diarrhea and ptosis may be mediated by mu2 and/or delta opioid receptor, while naloxone-precipitated jumping and body shakes may be mediated by mu1 opioid receptors.