Peptide Mimetic of BDNF Loop 4 Blocks Behavioral Signs of Morphine Withdrawal Syndrome and Prevents the Increase in ΔFosB Level in the Striatum of Rats.

Activity of compound GSB-106, a low-molecular mimetic of loop 4 of the brain neurotrophic factor (BDNF), was studied in experimental morphine withdrawal syndrome simulated in outbred rats. Single and subchronic (5 intraperitoneal injections) administration of GSB-106 in a dose of 0.1 mg/kg significantly reduced the total index of morphine withdrawal syndrome by 55.2 and 45.6%, respectively. GSB-106 reduced the severity of some behavioral signs (piloerection, gnashing of teeth, wet-dog shaking, and runaway attempts), but had no effect on mechanical allodynia formed in the rats with dependence. Subchronic treatment with GSB-106 prevented the increase in the content of ΔFosB (product of early response gene) in the striatum induced by morphine withdrawal. The results confirmed the concept on the involvement of neurotrophins, specifically BDNF and its analogs, in the mechanisms associated with the formation of opiate dependence.

Morphine Dependence is Attenuated by Treatment of 3,4,5-Trimethoxy Cinnamic Acid in Mice and Rats.

The effect of 3, 4, 5-trimethoxy cinnamic acid (TMCA) against morphine-induced dependence in mice and rats was investigated. Mice were pretreated with TMCA and then morphine was injected intraperitoneally; whereas rats were treated with TMCA (i.p.) and infused with morphine into the lateral ventricle of brain. Naloxone-induced morphine withdrawal syndrome and conditioned place preference test were performed. Moreover, western blotting and immunohistochemistry were used to measure protein expressions. Number of naloxone-precipitated jumps and conditioned place preference score in mice were attenuated by TMCA. Likewise, TMCA attenuated morphine dependent behavioral patterns such as diarrhea, grooming, penis licking, rearing, teeth chattering, and vocalization in rats. Moreover, the expression levels of pNR1and pERK in the frontal cortex of mice and cultured cortical neurons were diminished by TMCA. In the striatum, pERK expression was attenuated despite unaltered expression of pNR1 and NR1. Interestingly, morphine-induced elevations of FosB/ΔFosB+ cells were suppressed by TMCA (50, 100 mg/kg) in the nucleus accumbens sub-shell region of mice. In conclusion, TMCA could be considered as potential therapeutic agent against morphine-induced dependence.

Comparative Assessment of the Effectiveness of Noncompetitive NMDA Receptor Antagonists Amantadine and Hemantane in Morphine Withdrawal Syndrome Model.

Activities of noncompetitive NMDA receptor antagonists (aminoadamantane derivatives) were assessed in random-bred rats with modeled morphine withdrawal syndrome. A single intraperitoneal injection of hemantane (10 or 20 mg/kg) significantly and dose-dependently moderated some behavioral symptoms (teeth-chattering, ptosis, and vocalization) and reduced total score of morphine withdrawal syndrome. In morphine-abstinent rats, hemantane partially prevented the decrease in the thresholds of tactile sensitivity, but had no effect on locomotor activity and body weight loss. Under conditions of morphine withdrawal, intraperitoneal injection of amantadine (10 or 20 mg/kg) decreased motor activity and promoted body weight loss in parallel with the development of mechanical allodynia, but had no effect on the total withdrawal score. Comparison of aminoadamantane derivatives by behavioral and physiological parameters demonstrated the advantage of hemantane during morphine abstinence indicating the need of its study as a promising anti-addiction remedy.

Injection of Toll-like receptor 4 siRNA into the ventrolateral periaqueductal gray attenuates withdrawal syndrome in morphine-dependent rats.

We assessed the role of the Toll-like receptor 4 (TLR4) gene in the ventrolateral periaqueductal gray (vlPAG) region of morphine-dependent rats on attenuating withdrawal syndrome, and regulating glutamic acid decarboxylase (GAD67), glutamic acid (Glu), and gamma-aminobutyric acid (GABA). After siRNA-mediated downregulation of TLR4, changes were observed in withdrawal behavior and downstream signaling molecules. Rats were injected into the vlPAG with TLR4 siRNA, followed by intraperitoneal injection of morphine for 5 consecutive days, and then naloxone, and the behavioral indices of morphine withdrawal were observed. 'Wet-dog' shakes, teeth chattering, and the total scores of withdrawal reactions were reduced. TLR4 expression and Glu levels were reduced, whereas GAD67 and GABA levels were increased. Overall, these findings indicate that modifying TLR4 gene expression in the vlPAG stimulates expression of the downstream signaling molecule, GAD67, which decreases Glu levels and increases GABA levels. This mechanism may explain the inhibition of withdrawal syndrome in morphine-dependent rats.

Chromium picolinate reduces morphine-dependence in rats, while increasing brain serotonin levels.

Chromium is an essential trace element with anti-diabetic and anti-depressant effect; the latter is considered related to chromium properties of increasing brain serotonin. Cr3+ salts were shown to improve some forced swimming-parameters and to induce rewarding effects, which are additive to those of morphine, but Cr effect on addictive processes has not been tested. AIM: The present study aimed to assess chromium picolinate (CrPi) influence on morphine-dependence in rats. MATHERIAL AND METHODS: We used five groups of 10 rats. Groups 1 and 2 (controls) received saline, respectively CrPi, 0.01 mg/kg/day, for 10 days. In groups 3, 4 and 5 dependence was induced with progressively-increased morphine doses (from 5 - day 1-90 mg/kg/day - day 10, s.c.). Group 3 received only morphine, while groups 4 and 5 received CrPi, i.p., 10 and respectively 5 µg/kg/day, during the 10 days of dependence induction. On day 11, groups 3, 4, and 5 were administered 90 mg/kg morphine, and, 2 h later, all rats received naloxone, 2 mg/kg s.c., to precipitate withdrawal. We compared withdrawal intensity in group 3 vs. groups 4 and 5, assessing both individual symptoms and Gellert-Holtzman global withdrawal score. Upon rats sacrifice at the end of the experiments, brain serotonin (5HT) in certain areas and serum Cr were assessed. RESULTS: Some withdrawal signs were unequally influenced by CrPi: compulsive mastication was reduced by both CrPi doses (p < 0.05), while teeth chattering and grooming were significantly reduced only by the higher dose (p < 0.05). Withdrawal score was reduced by both CrPi doses: from 132.4 ±â€¯9.87 - group 3 to 122.2 ±â€¯6.47 - group 4 (p < 0.01 vs. group 3) and 124.1 ±â€¯8.41 - group 5 (p < 0.05 vs. group 3). CrPi reduction of withdrawal is accompanied by increased brain 5 H T, mainly in the prefrontal cortex (646.3 ±â€¯8.51 - group 3 vs. 661.5 ±â€¯14.63 - group 4, p < 0.01 and 660.7 ±â€¯14.01 pg/mg tissue - group 5, p < 0.05 vs. group 3). CrPi also increases brain 5 H T in non-dependent rats (prefrontal cortex: 541.6 ±â€¯31.80, group 1 and 565.5 ±â€¯16. 46 pg/mg tissue, group 2, p < 0.05). Administration of CrPi determined a dose-dependent increase of serum Cr. CONCLUSIONS: Our study evidenced a slight, but significant reduction of morphine dependence in rats induced by chromium picolinate, accompanied by increased brain serotonin. This might be considered a supplementary evidence for chromium anti-depressant effect and its serotonin-mediated mechanisms.

Behavioral expression of opiate withdrawal is altered after prefrontocortical dopamine depletion in rats: monoaminergic correlates.

The objective of this study was to establish the effects of prefrontocortical dopamine depletion on opiate withdrawal and prefrontocortical neurochemical changes elicited by morphine dependence and withdrawal. The dopaminergic content was also measured in the nucleus accumbens during withdrawal, in order to detect reactive changes induced by prefrontocortical lesion. Withdrawal was induced by naloxone in morphine-dependent rats. Monoamine levels were analyzed post-mortem by high performance liquid cromatography. The results showed that chronic morphine dependence did not modify basal levels of monoamines in sham rats, revealing neuroadaptation of prefrontocortical dopamine, noradrenaline and serotonin systems to chronic morphine. The neuroadaptive phenomenon remained after prefrontocortical lesion (> 79% dopamine depletion). On the other hand, a strong increase of dopamine, noradrenaline, and serotonin contents in the medial prefrontal cortex of sham rats was detected during opiate withdrawal. However, in lesioned rats, the increase of prefrontocortical dopamine and serotonin content, but not that of noradrenaline, was much lower. In the nucleus accumbens, prefrontocortical lesion reactively enhanced the dopaminergic tone and, although opiate withdrawal reduced dopaminergic activity in both sham and lesioned rats, this reduction was less intense in the latter group. At a behavioral level, some symptoms of physical opiate withdrawal were exacerbated in lesioned rats (writhing, mastication, teeth-chattering, global score) and exploration was reduced. The findings hence indicate that: (i) prefrontocortical monoaminergic changes play a role in the behavioral expression of opiate withdrawal; (ii) the severity of some withdrawal signs are related to the dopaminergic and serotonergic tone of the medial prefrontal cortex rather than to the noradrenergic one, and (iii) an inverse relationship between mesocortical and mesolimbic dopaminergic systems exists.

Competitive and glycine/NMDA receptor antagonists attenuate withdrawal-induced behaviours and increased hippocampal acetylcholine efflux in morphine-dependent rats.

The present study has examined the glycine/N-methyl-D-aspartate (NMDA) receptor antagonist, R-(+)-3-amino-1-hydroxypyrrolid-2-one (R-(+)-HA-966) and the competitive NMDA receptor antagonist, cis-4-(phosphonomethyl)piperidine-2-carboxylic acid (CGS 19755) on the behavioural syndrome and increased hipppocampal acetylcholine efflux induced during morphine-withdrawal in the rat. Subcutaneous naltrexone (1 mg/kg) injection, 48 hr after implantation of a 75 mg morphine pellet, induced a robust withdrawal syndrome consisting of wet dog shakes, ejaculations, mouth movement, ptosis, irritability to touch and diarrhoea. Pretreatment with the alpha2-adrenoceptor agonist, clonidine (0.1-0.4 mg/kg), R-(+)-HA-966 (10-60 mg/kg) or CGS 19755 (5 or 10 mg/kg) significantly reduced the incidence of withdrawal behaviours. In addition, all three compounds significantly attenuated the increase in hippocampal acetylcholine efflux induced following naltrexone (1 mg/kg, s.c.) injection in morphine-dependent rats. These results provide further evidence demonstrating that NMDA receptor antagonists attenuate both the behavioural and neurochemical effects observed during morphine withdrawal in the rat.

Alterations of adenosine A1 receptors in morphine dependence.

The possibility that central adenosine A1 and A2a receptors mediate opiate dependence was examined in morphine-treated mice using radioligand binding methods. Mice treated with morphine for 72 h demonstrated significant increases in naloxone precipitated abstinence behaviors of jumping, wet-dog shakes, teeth chattering, forepaw trends, forepaw tremors and diarrhea compared to vehicle-treated mice. Increased concentrations of cortical adenosine A1 receptor sites, but not striatal adenosine A2a sites, were found in saturation binding studies from morphine-dependent mice. Decreases in cortical A1 agonist binding affinity values along with increases in agonist binding sites were demonstrated in competition binding studies. These results suggest that adaptive changes of upregulation and sensitization of adenosine A1 receptors play a role in mediating the opiate abstinence syndrome.

Withdrawal and bidirectional cross-withdrawal responses in rats treated with adenosine agonists and morphine.

The aim of this study was to investigate whether the A1/A2 receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), and the selective A1 agonist, N6-cyclopentyladenosine (CPA), induced physical dependence by quantifying specific antagonist-precipitated withdrawal syndromes in conscious rats. In addition, the presence of bidirectional cross-withdrawal was also investigated. The agonists were administered s.c. to groups of rats at 12 h intervals. Antagonists were administered s.c., 12 hours after the last dose, followed by observation and measurement of faecal output for 20 min. NECA (4 x 0.03 mg kg(-1), s.c) and CPA (4 x 0.03, 0.1 and 0.3 mg kg(-1), s.c.) induced physical dependence, as shown by the expression of a significant withdrawal syndrome when challenged with the adenosine A1/A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX, 0.1 mg kg(-1), s.c.) and the A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPDPX, 0.1 mg kg(-1), s.c.) respectively. The syndromes consisted of teeth chattering and shaking behaviours shown to occur in morphine-dependent animals withdrawn with naloxone viz, paw, body and 'wet-dog' shakes, but with the additional behaviours of head shaking and yawning. In further contrast to the opiate withdrawal syndrome, no diarrhoea occurred in the groups of animals treated with adenosine agonists and withdrawn with their respective antagonists. Bidirectional cross-withdrawal syndromes were also revealed when naloxone (3 mg kg(-1), s.c.) was administered to adenosine agonist pre-treated rats and adenosine antagonists were given to morphine pre-treated rats. This study provides further information illustrating that close links exist between the adenosine and opiate systems.

Inhibitory effect of the NMDA receptor antagonist, dizocilpine (MK-801), on the development of morphine dependence.

We investigated the effect of a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imin ehydrogen maleate (dizocilpine, MK-801), on hippocampal norepinephrine release in morphine-treated rats in order to clarify the relationship between NMDA receptors and the development of morphine dependence. Naloxone hydrochloride injected subcutaneously (s.c.) into morphine-dependent rats, induced an immediate increase in hippocampal norepinephrine release, which was associated with a typical morphine withdrawal syndrome. The increased norepinephrine levels persisted for at least 2 hr, even after the disappearance of the behavioral withdrawal syndrome. This striking effect of naloxone on hippocampal norepinephrine release was dependent on the duration of the intracerebroventricular (i.c.v.) morphine infusion. Pretreatment with dizocilpine (s.c.) before naloxone challenge reduce the rate of the rise in hippocampal norepinephrine release induced by naloxone in morphine-treated rats. Concurrent infusion (i.c.v.) of dizocilpine and morphine decreased the level of hippocampal norepinephrine release after a naloxone challenge. Both pretreatment with dizocilpine (s.c.) before naxolone injection and infusion (i.c.v.) of dizocilpine suppressed rearing and teeth-chattering signs, but not wet-dog shakes in morphine-treated rats. These results suggest that dizocilpine attenuates the development of morphine dependence through NMDA receptors, and thus that interaction between opioid receptors and NMDA receptors may be involved in the development of morphine dependence.

Blockade of orexin type-1 receptors in locus coeruleus nucleus attenuates the development of morphine dependency in rats.

The aim of this study was to evaluate the effects of orexin type-1 receptor (OX1R) antagonism in locus coeruleus (LC) nucleus on the development of morphine physical dependence in rats. Animals were rendered dependent on morphine by subcutaneous (s.c.) administration of morphine sulfate (6, 16, 26, 36, 46, 56 and 66 mg/kg, 2 ml/kg) at set intervals of 24 h for 7 days. Immediately before each morphine administration, the animals received intra-LC administration of SB-334867 (3 mM, 0.2 µl), a selective orexin type-1 receptor antagonist. On day 8, naloxone (3 mg/kg, i.p.) was injected and physical dependence was evaluated for 30 min. Our results showed that administration of OX1R antagonist before each morphine injection significantly decreased somatic signs of naloxone-induced morphine withdrawal syndrome, including defecation, wet-dog shake, diarrhea, jumping, scratching, and teeth chattering. These results suggest that the activation of OX1R in LC nucleus might be involved in the development of morphine dependency.

Antagonism of orexin type 1 receptors in the locus coeruleus attenuates signs of naloxone-precipitated morphine withdrawal in rats.

It has been shown that orexin neuropeptides contribute to morphine-induced physical dependence. The locus coeruleus (LC), which receives a dense extra-hypothalamic orexinergic projection, is a key brain region implicated in the expression of somatic signs of morphine withdrawal syndrome. The aim of the present study is to investigate the role of LC orexin type 1 receptors (OXR1) on naloxone-precipitated morphine withdrawal signs in rats. Adult male Wistar rats were rendered dependent on morphine by subcutaneous (s.c.) injection of morphine sulfate (10mg/kg) at an interval of 12h for 9 days. On day 10, naloxone (1mg/kg i.p.) was injected 2h after morphine administration. Somatic signs of withdrawal were then evaluated in a clear Plexiglas test chamber (30 cm diameter, 50 cm height) for 25 min. One group of animals received intra-LC SB-334867-A, a selective OXR1 antagonist, (100 microM, 0.2 microl) immediately before naloxone. In the control group, SB-334867-A vehicle was microinjected into the LC in the same manner. The results showed that intra-LC OXR1 receptor blockade significantly decreased the somatic signs of withdrawal including chewing, diarrhea, scratching, teeth chattering, wet-dog shake and ptosis. These results suggest that activation of OXR1 in the LC might be involved in the expression of withdrawal signs in morphine dependent rats.

The effects of different 4-aminopyridine and morphine combinations on the intensity of morphine abstinence.

The glutamatergic system is deeply involved in the development of opiate dependence and in the manifestation of opiate abstinence syndrome. In this study the effect of the increase in the endogenous glutamate (GLU) release due to 4-aminopyridine (4-AP), a potassium channel blocker, during the development of morphine (M) physical dependence and during the naloxone (NL)-precipitated abstinence syndrome was investigated. For the development of physical dependence M was intraperitoneally (i.p.) injected for 9 days 105 min following i.p. saline administration to a group of rats. In the first 3 days the dose of M was 10 mg x kg(-1). In the second 3 days the initial dose was doubled (20 mg x kg(-1)) and in the last 3 days the dose of M was raised to 40 mg x kg(-1). On day 10, the rats were divided into three groups at random and these three groups were i.p. given saline 105 min before 80 mg x kg(-1)M, 2 mg x kg(-1) 4-AP 105 min after 80 mg x kg(-1) M, and 80 mg x kg(-1) M 105 min before 2 mg x kg(-1) 4-AP, respectively. In a second group of rats, the rats were i.p. given 2 mg x kg(-1) 4-AP 105 min prior to M administration, which was increased every 3 days (10 mg x kg(-1), 20 mg x kg(-1), 40 mg x kg(-1)). On day 10, the rats were divided into two groups whose first injection was saline and 2 mg x kg(-1) 4-AP, respectively. The second injections of both groups after an interval of 105 min following the first one contained 80 mg x kg(-1) M. In contrast, one group of rats received only i.p. saline at every other injection time (the control group). Furthermore, another group of rats was i.p. administered 2 mg x kg(-1) 4-AP once a day, as the first injection. At the second injection time they were i.p. given saline. After a period of 15 min following the last administration on day 10, the rats belonging to all groups were i.p. injected with 2 mg x kg(-1) NL and immediately placed in a metal cage. Body weight loss (g), teeth chattering, rearing, wet-dog shaking, grooming, and jumping were determined or counted for 15 min. Penile erection, defecation, and diarrhoea were separately scored with one point for every individual occurrence, and the total score was named 'total number of others'. The administration of 4-AP before M appeared to intensify the development of dependence, and was most probably due to the Ca2+-induced inactivation of NMDA receptors as a result of excess release of GLU when the 4-AP took effect. The inactivation of NMDA receptors should have acted as a transient blockade of the receptors during the chronic administration period, and as well as after a single administration on day 10 before M injection and before abstinence. The intensification of the abstinence syndrome may be dependent on the excessive GLU released by 4-AP.

Effect of nimodipine, diltiazem and BAY K 8644 on the behavioural and neurochemical changes associated with naloxone-precipitated withdrawal in the rat. A comparison with clonidine.

1. Nimodipine, diltiazem and BAY K 8644 decreased the incidence of wet dog shakes, teeth chattering, grooming and diarrhoea to a similar degree of clonidine. 2. Nimodipine, diltiazem and BAY K 8644 had no effect on changes in the serotonin metabolism induced by naloxone precipitated abstinence syndrome. 3. Clonidine decreased the ratio of serotonin turnover in the brain of morphine-dependent rats. 4. From these experiments it is concluded that nimodipine, diltiazem and BAY K 8644 exert their effects in preventing morphine withdrawal symptoms through a mechanism independent of the serotonergic system.

Effects of peptidase inhibitors, [D-Ala2, Met5]-enkephalinamide and antiserum to methionine-enkephalin microinjected into the caudal periaqueductal gray on morphine withdrawal in rats.

We examined the involvement of enkephalins in the caudal periaqueductal gray (cPAG) in morphine withdrawal in rats. Rats were treated with increasing doses of morphine (20-30 mg/kg/day, s.c., for 5 days) to develop morphine dependence. Morphine withdrawal was induced by naloxone (5 mg/kg, s.c.) 24 hr after the final morphine injection. The level of preproenkephalin (PPE) mRNA in the cPAG was estimated by quantitative in situ hybridization. PPE mRNA in the cPAG was increased 4-24 hr after naloxone in morphine-treated rats. A mixture of peptidase inhibitors (0.5 microl of a solution of amastatin, captopril and phosphoramidon, 3 x 10(-3) M each) microinjected into the cPAG suppressed morphine withdrawal (a decrease in the number of jumping, chin rubbing, paw rubbing and teeth chattering). Antiserum to methionine-enkephalin (1:10 dilution) microinjected into the cPAG did not significantly aggravate morphine withdrawal with or without the mixture of peptidase inhibitors. However, [D-Ala2, Met5]-enkephalinamide (20 nmol), an enkephalin analog, injected into the cPAG decreased the number of jumping without any influence on the other withdrawal signs. These results suggest that the increase in enkephalins in the cPAG may participate in the alleviation of morphine withdrawal (jumping behavior).