Dopamine D1 receptor-dependent modifications in the dopamine and cAMP-regulated phosphoprotein of Mr 32 kDa phosphorylation pattern in striatal areas of morphine-sensitized rats.

Morphine sensitization is a model of latent, functionally inducible increase in dopamine D(1) receptor-mediated transmission, which may be unmasked by an external stimulus. Morphine-sensitized rats present dopamine D(1) receptor-dependent stereotypies upon morphine challenge and resilience to unavoidable stress-induced behavioral deficits. This tonic increase in dopamine D(1) dopaminergic transmission is counter-adaptive to an enhanced mu-opioid receptor-dependent signaling in striatal areas. Control and sensitized rats show a similar dopamine and cAMP-regulated phosphoprotein of M(r) 32 kDa (DARPP-32) phosphorylation pattern in striatal areas. Acute morphine administration induced an early increase and delayed decrease in phospho-threonine (Thr)34 DARPP-32 levels accompanied by a delayed increase in phospho-Thr75 DARPP-32 levels in the nucleus accumbens and caudate-putamen of sensitized rats, while it had no effects in control animals. The administration of a selective dopamine D(1) receptor antagonist (SCH 23390) before morphine challenge prevented the behavioral and neurochemical modifications in sensitized rats. 6-Methyl-2-(phenylethynyl)-pyridine, a selective metabotropic glutamate receptor 5 (mGluR(5)) antagonist, administered 1 h after morphine challenge, prevented the delayed phosphorylation changes, but it had no effect when administered before challenge. Moreover, the DARPP-32 phosphorylation pattern in the caudate-putamen of sensitized rats after unavoidable stress exposure was studied. The stress-induced neurochemical modifications and their sensitivity to receptor antagonists were similar to those observed after acute morphine administration. In conclusion, these results suggest that in the experimental conditions used an increase in dopamine output in striatal areas is followed by a complex neurochemical pattern, in which the initial stimulation of dopamine D(1) receptors triggers a sequence of signaling events that lead to an mGluR(5)-mediated increase in phospho-Thr75 DARPP-32 levels. Since DARPP-32 phosphorylated in Thr75 inhibits cAMP-dependent protein kinase (PKA) activity, the final result is a decrease in the dopamine D(1) receptor-dependent phosphorylation events.

Dopamine and cyclic AMP-regulated phosphoprotein-32 phosphorylation pattern in cocaine and morphine-sensitized rats.

This study reports some of the modifications in dopaminergic signalling that accompany cocaine and morphine behavioural sensitization. Cocaine-sensitized rats showed increased phosphorylation of dopamine- and cyclic AMP-regulated phosphoprotein Mr 32 kDa (DARPP-32) at threonine-75 (Thr75) and decreased DARPP-32 phosphorylation at Thr34, in the caudate-putamen (CPu) and nucleus accumbens (NAc) 7 days after sensitization assessment. Conversely, in morphine-sensitized rats, no apparent modifications in DARPP-32 phosphorylation pattern were observed. Morphine-sensitized rats have increased binding and coupling of micro -opioid receptors and increased dopaminergic transmission in striatal areas and, upon morphine challenge, exhibit dopamine D1 receptor-dependent stereotypies. Thus, the DARPP-32 phosphorylation pattern was studied in morphine-sensitized rats at different times after morphine challenge. Morphine challenge increased levels of phospho-Thr75 DARPP-32 and decreased levels of phospho-Thr34 DARPP-32 in a time-dependent manner in the CPu and NAc. In order to assess whether these modifications were related to modified cyclic AMP-dependent protein kinase (PKA) activity, the phosphorylation levels of two other PKA substrates were examined, the GluR1 and NR1 subunits of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and NMDA receptors respectively. The phosphorylation levels of GluR1 and NR1 subunits decreased in parallel with those of phospho-Thr-34 DARPP-32, supporting the hypothesis that morphine challenge elicited a decrease in PKA activity in morphine-sensitized rats.

Acquisition of a palatable-food-sustained appetitive behavior in satiated rats is dependent on the dopaminergic response to this food in limbic areas.

Rats exposed to repeated unavoidable stress show decreased dopamine output in the nucleus accumbens shell (NAcS) and do not acquire vanilla sugar (VS)-sustained appetitive behavior (VAB). Rats treated with lithium for 3 weeks also show decreased NAcS dopamine output, yet they acquire VAB. Feeding a novel palatable food increases extraneuronal dopamine levels in the NAcS and medial prefrontal cortex (mPFC) in rats. In order to investigate the role of food-induced dopamine release in VAB acquisition, we studied by microdialysis the dopaminergic response in the NAcS and mPFC to the presentation and consumption of VS in satiated control rats, and in satiated rats exposed to repeated stress or lithium treatment. The dopaminergic response to VS was also studied in rats familiar with VS, or that had acquired VAB. In control rats, VS feeding was accompanied by increased dopamine output in the NAcS and mPFC, and one-trial habituation to this effect developed in the NAcS. Rats exposed to a 7-day stress showed reduced interest in VS pellets, and when fed VS they did not show a dopaminergic response in the NAcS and mPFC. Lithium-treated rats rapidly ate VS pellets and showed increased dopamine output in the NAcS and mPFC, with no habituation in the NAcS response. Rats familiar with VS and rats that had already learned VAB ate VS pellets. The first group showed a lower dopaminergic response to VS consumption than the control group, but the latter showed no dopaminergic response in the NAcS and mPFC. We propose that the limbic dopaminergic response to a novel palatable food plays a role in associative learning and that it is predictive of the competence to learn an appetitive behavior. Moreover, in our experimental conditions a phasic increase in mesolimbic dopamine no longer signals the VS stimulus once it has become a reinforcer in an appetitive task.

Dizocilpine infusion has a different effect in the development of morphine and cocaine sensitization: behavioral and neurochemical aspects.

The stimulation of glutamate receptors plays a relevant role in the development of behavioral sensitization to psychostimulants, while less clear results have been obtained on their role in morphine sensitization. We addressed this issue by comparing the development of cocaine and morphine sensitization under a continuous s.c. infusion of the N-methyl-D-aspartate (NMDA) antagonist dizocilpine (0.1 mg/kg/24 h). Moreover, we studied the expression of NMDA and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits in discrete limbic areas of rats sensitized to morphine or cocaine with or without the concomitant dizocilpine infusion. It was observed that dizocilpine infusion did not prevent the development of morphine sensitization, while it prevented the development of tolerance to morphine-induced analgesia. Finally, morphine-sensitized animals did not present any modification in the subunit expression of glutamate receptors in the brain areas examined. In agreement with previous results, we found that dizocilpine infusion prevented the development of cocaine sensitization. Moreover, we observed that rats sensitized to cocaine presented a significant increase in the levels of GLUR1, NR1 and NR2B, in the nucleus accumbens, and of NR2B in the hippocampus compared to control animals. Such modifications were absent in rats administered cocaine under dizocilpine infusion. We conclude that: (i) morphine sensitization is a neuroadaptive phenomenon which does not appear to require NMDA receptor activity in order to develop; (ii) cocaine sensitization is clearly dependent on NMDA receptor activity, as dizocilpine infusion prevented the occurrence of glutamate receptors modifications as well as the development of sensitization.

Long-term therapy using GHB (sodium gamma hydroxybutyrate) for treatment-resistant chronic alcoholics.

Thirty-five alcohol-dependent patients according to DSM-IV criteria who also met criteria for treatment resistance were treated with doses of gamma hydroxybutyrate (GHB) ranging between 25 and 100 mg/kg/die in an open one-year study. The results show that no patients discontinued the program during the first month of treatment. Sixty percent of these patients successfully completed the protocol; 11.4% showed complete abstinence (full responder patients); 14.3% strongly reduced their alcohol intake (partial responder patients) and 34.3% of the patients were still under treatment after one year. Forty percent of the patients were nonresponders. The retention rate under treatment of the studied sample was statistically higher than that found during the last treatment of the same subjects. No significant differences were found between full responder and partial responder patients regarding changes in clinical features, alcohol intake or social adjustment. Patients still in treatment after one year significantly differed from nonresponder patients on all the variables investigated. A six-times/daily fractionated administration of the GHB dose was the only significant predictor of the retention rate.

Dopamine output in the nucleus accumbens shell is related to the acquisition and the retention of a motivated appetitive behavior in rats.

Chronic stress exposure consistently impairs the reactivity to aversive and pleasurable stimuli in rats; these behavioral modifications are associated with a decrease in dopamine output in the nucleus accumbens shell (NAcS). However, when rats that have already acquired an appetitive behavior are exposed to chronic stress, they develop an impaired reactivity to avoidable aversive stimuli while retaining the appetitive behavior. The dissociation between these two behavioral traits was used to study whether the decreased dopaminergic activity in the NAcS was connected to either of the two deficits. Dopamine output was studied through microdialysis as dopamine accumulation following re-uptake inhibition by cocaine. When rats that had previously acquired the appetitive behavior were exposed to chronic stress, they showed a dopaminergic transmission in the NAcS similar to that of controls and significantly higher than that of chronically stressed animals. Thus, dopamine output in the NAcS was consistently associated to the acquisition and maintenance of appetitive behavior, while the expression of a deficit in avoidance appeared to be independent of it.

Animal models for the study of antidepressant activity.

Three behavioral paradigms are presented for the study of the mechanism of action of antidepressant treatments and for the screening of new antidepressant drugs. The first model (acute escape deficit) exploits the decreased ability of a rat exposed to an unavoidable stress to avoid a noxious stimulus, and it allows us to evaluate the preventive activity of a treatment on the development of escape deficit. The second paradigm (chronic escape deficit) begins as acute escape deficit, that is then indefinitely sustained by the repeated administration of mild stressors; this model allows us to evaluate the efficacy of a treatment to revert the escape deficit. The third is a model of anhedonia based on the finding that exposure to repeated unavoidable stress prevents the acquisition of an appetitive behavior induced and maintained by a highly palatable food (vanilla sugar) in rats fed ad libitum; this paradigm assesses the efficacy of a treatment to restore an animal's motivation. A long-term (2 to 3 week) treatment with classical antidepressants, such as imipramine or fluoxetine, resulted in a clear-cut preventive and/or revertant activity in the three models.

Intensive treatment with lithium carbonate "once a day" in bipolar patients.

Lithium carbonate is the treatment of choice in the prevention of recurrences of bipolar mood disorders. The aim of this study is the evaluation of the pharmacokinetics of a new lithium compound to be administered "once a day", with a half-life of 32.4 hours. The present study investigates plasma lithium levels in patients treated with the novel vs. standard preparation of lithium carbonate to assess whether the new preparation may be associated with lithium plasma accumulation. The authors studied twenty-two bipolar patients whose standard lithium treatment was replaced by the new one. The total duration of the new treatment was 57 days. During this period, lithium plasma levels were monitored 7 times, 12 hours post-lithium administration. Lithium plasma level 24 hours following administration was evaluated in seven patients. The average dose of "once a day" lithium was 821 (+/- 250) mg/day, the corresponding plasma level was 0.45 (+/- 0.11) mEq/L, and the dose (g) to plasma level ratio was 1.96 (+/- 0.66). Differences between average lithium plasma levels at time T1 and successive times were not statistically significantly. Moreover, the average lithium level at 12 hours after lithium administration was not different from that after 24 hours (0.45 +/- 0.11 and 0.47 +/- 0.0.29 respectively). These results demonstrate that the new preparation, "once a day", maintains steady lithium plasma levels during our times of observation without accumulation in body fluids and tissues. "Once a day" lithium carbonate appears to be a useful alternative to the standard preparation, and may potentially improve treatment compliance by simplifying patients' medication.

Acquisition of an appetitive behavior reverses the effects of long-term treatment with lithium in rats.

Rats exposed to a long-term treatment with lithium chloride develop a deficit of avoidance accompanied by a reduction in the basal levels of extraneuronal dopamine and in dopamine accumulation in the nucleus accumbens shell after acute uptake inhibition. Such a condition is similar to that of an experimental model of depression induced by exposing rats to a chronic stress procedure. Rats exposed to chronic stress are also unable to acquire an appetitive behavior sustained by a highly palatable food. Thus, it was studied whether rats fed a diet containing lithium would develop an appetitive behavior induced by a pure hedonic stimulus. Rats on the lithium diet developed a clear-cut escape deficit condition accompanied by a decreased dopamine output in the nucleus accumbens shell; nevertheless, they learned the appetitive behavior within a similar period to controls. The development of the appetitive behavior coincided with the recovery of the capacity to avoid a noxious stimulus and with the return of the dopaminergic transmission in the nucleus accumbens shell to values similar to those of control rats. It may be concluded that the mechanism of action underlying the behavioral and neurochemical sequelae of a chronic stress is distinct from that of the analogous effects produced by lithium.

Long-term lithium administration abolishes the resistance to stress in rats sensitized to morphine.

Morphine sensitized rats appear protected from the sequelae of an unavoidable stress: when exposed to stress (after a 7-day morphine wash-out) and then tested for escape, they perform like naive animals. This protection appears similar to that induced by chronic imipramine treatment, as it is antagonized by the inhibition of D(1)-dopamine receptors before exposure to unavoidable stress. Repeated unavoidable stress induces in rats a condition characterized by hyporeactivity to noxious stimuli and reverted by long-term antidepressant treatments, and this state is regarded as an experimental model of depression. The resistance to stress in morphine sensitized rats could be considered as the behavioral counterpart of the sensitivity to stress in control rats, i.e. as a model of mania. The aim of the present study was to validate such a putative model by studying whether the resistance to stress induced by morphine sensitization would respond to a long-term administration of lithium, the reference antimanic drug. Long-term lithium treatment induces in rats a condition of hyporeactivity to noxious stimuli, accompanied by decreased levels of dopamine in the nucleus accumbens shell. In morphine sensitized rats chronic lithium abolished the resistance to stress, but it did not modify the D(1)-dopamine receptor mediated response to morphine, nor did it modify the levels of extraneuronal dopamine in the nucleus accumbens shell. Thus, lithium treatment abolished the resistance to stress in morphine sensitized rats, conferring predictive validity to the paradigm. Moreover, it did so through a mechanism which appeared to be independent of D(1)-dopamine receptor activity.

Buprenorphine: a controlled clinical trial in the treatment of opioid dependence.

Clinical trials carried out to compare methadone and buprenorphine in the treatment of opioid dependence have generally employed an alcoholic solution of buprenorphine, which has a bioavailability superior to that of the tablets. Since the product available for large scale use is in tablet form, one intended to verify the efficacy of this formulation. In a multicentre randomised controlled double blind study, 72 opioid dependent patients were assigned to treatment with buprenorphine (8 mg/day) or methadone (60 mg/day) for a period of 6 months. The two compounds did not show any significant difference with regard to urinalyses: the average percentage of analyses proving negative was 60.4% for patients assigned to buprenorphine, and 65.5% for those assigned to methadone. With regard to retention, a non-significant trend in favour of methadone was observed. Patients completing the trial improved significantly in terms of psychosocial adjustment and global functioning, as ascertained by the DSM-IV-GAF and symptom checklist-90 (SCL-90) scales, and this was independent of the treatment group. Finally, in the case of buprenorphine, patients who dropped out differed significantly from those who stayed, in terms of a higher level of psychopathological symptoms, and a lower level of psychosocial functioning. The results of the study further support the utility of buprenorphine for the treatment of opioid dependence.

Methadone dose and retention during treatment of heroin addicts with Axis I psychiatric comorbidity.

We studied 90 opioid-dependent subjects, 38 with one or more additional Axis I diagnosis and 52 with no psychiatric comorbidity. There were significant differences between these two groups regarding the methadone dose required for clinical stabilization, but not in the rate of retention in treatment. Dual Diagnosis patients, those with psychiatric comorbidity, required an average stabilization dose of 154 +/- 84 of methadone compared to 99 +/- 49 mg/day for patients whose only Axis I diagnosis was Opioid Dependence. In the 990-day period considered there were no differences between the two groups of patients in terms of retention in treatment.

Rats sensitized to morphine are resistant to the behavioral effects of an unavoidable stress.

In agreement with the results of other authors, rats sensitized to morphine and challenged with 5 mg/kg of morphine after 7 days of wash-out showed intense stereotyped movements, the expression of which was selectively antagonized by SCH 23390. Sensitized rats were exposed to an unavoidable stress (which consistently produces an escape deficit in control animals) after 3, 7 and 21 days of morphine wash-out. Twenty-four hours after the unavoidable stress, animals were tested for their capacity to escape and their performance was compared to that of control-stressed and naive rats. Morphine sensitization completely prevented the development of escape deficit. This protective effect was similar to that induced by a chronic imipramine treatment and, like the effect of imipramine, it was antagonized by the administration of SCH 23390 before the unavoidable stress. However, it was not affected by the administration of naloxone. Moreover, when rats presenting a clear-cut escape deficit, induced by a 10-day treatment with SKF 38393, were exposed to the morphine sensitization protocol, a complete recovery of their capacity to avoid a noxious stimulus was observed. Finally, the down-regulation of both the number of D(1)-dopamine receptors and of the coupled adenylyl cyclase activity in the pre-frontal cortex (PFC) produced by long-term SKF 38393 administration was reverted by the superimposed morphine sensitization. Thus, the condition of morphine sensitization appears to share several common effects with chronic imipramine treatment.

Efficacy of an Hypericum perforatum (St. John's wort) extract in preventing and reverting a condition of escape deficit in rats.

The treatment of unselected depressed patients with an hydro-alcoholic extract of Hypericum perforatum has been reported to have an efficacy similar to that of classical antidepressants. In the present report, the effects of H. perforatum were studied on three animal models of depression: (i) an acute form of escape deficit (ED) induced by an unavoidable stress; (ii) a chronic model of ED, which can be maintained by the administration of mild stressors on alternate days; (iii) a model of anhedonia based on the finding that repeated stressors prevent the development of an appetitive behavior induced by vanilla sugar in satiated rats fed ad libitum. H. perforatum acutely protected animals from the sequelae of unavoidable stress; such an effect was partially prevented by the administration of SCH 23390 or (-)-pindolol. Moreover, H. perforatum reverted the ED maintained by repeated stressors and preserved the animal's capacity to learn to operate for earning a positive reinforcer. It was concluded that H. perforatum contains some active principle(s) endowed with antidepressant activity.

The effects of long-term administration of rubidium or lithium on reactivity to stress and on dopamine output in the nucleus accumbens in rats.

Rubidium and lithium are alkali metals belonging to the same periodic series as sodium, potassium and cesium. In the present report the effects of lithium and rubidium on animal reactivity to stressful stimuli and on dopamine output in the nucleus accumbens were studied. A dose-response curve with rubidium, administered acutely before exposure to unavoidable stress, showed a maximal protective activity on escape deficit development at the dose of 0. 41 mEq/kg. Rubidium injected at doses of 0.008-0.08 mEq/kg 72 h before the unavoidable stress had the same efficacy as the acute 0. 41 mEq/kg dose. Tolerance to the effect of rubidium developed after 9 days of treatment and, on day 15, rats presented a spontaneous escape deficit. The acute effect of lithium, administered for 3.5 days at the dose of 0.8 mEq/kg, i.p. twice a day before the exposure to unavoidable stress, was analogous to that of rubidium, but after repeated treatment a spontaneous escape deficit developed. Rats showing an escape deficit secondary to chronic stress also presented decreased extraneuronal dopamine concentrations in the nucleus accumbens. Accordingly, microdialysis studies showed significantly lower extracellular dopamine levels in rats chronically treated with lithium or rubidium compared to control animals. Cocaine (5 mg/kg i. p.) administered acutely increased extracellular dopamine concentrations in control rats, as well as in rats chronically stressed or chronically treated with lithium or rubidium. However, the dopamine increase was significantly higher in controls compared to the other groups. In conclusion, long-term treatment with lithium or rubidium, or the exposure to chronic stress, produced a condition of behavioral hypo-reactivity accompanied by a decreased dopamine output in the nucleus accumbens.

A chronic stress that impairs reactivity in rats also decreases dopaminergic transmission in the nucleus accumbens: a microdialysis study.

Chronic stress induces in rats a decreased reactivity toward noxious stimuli (escape deficit), which can be reverted by antidepressant treatments. The present study reports that this condition of behavioral deficit is accompanied by a decreased level of extracellular dopamine in the nucleus accumbens shell. To assess whether this finding was the result of a decreased release or of an enhanced removal of dopamine, we acutely administered cocaine, and 2 h later d-amphetamine, to stressed and control rats. The increases in dopamine output observed in stressed animals after cocaine administration were significantly lower than those observed in control rats; whereas the total amount of dopamine released after d-amphetamine administration was similar in both groups of rats. These data suggest a reduced activity of dopaminergic neurons as the possible mechanism underlying dopamine basal level reduction in stressed animals. It is interesting that the stress group showed a locomotor response to cocaine not different from control rats, thus suggesting a condition of sensitization to dopamine receptor stimulation. Imipramine administered daily concomitantly with stress exposure completely reverted the escape deficit condition of chronically stressed rats. Moreover, stressed rats treated with imipramine showed basal and cocaine stimulated levels of extraneuronal dopamine similar to those observed in control animals.

Under continuous dizocilpine infusion an N-methyl-D-aspartate receptor independent form of cocaine sensitization develops in rats.

N-methyl-D-aspartate (NMDA) receptor blockade is thought to prevent the development of cocaine-induced sensitization. Moreover, when cocaine is administered daily along with dizocilpine infusion to previously sensitized rats, the extinction of sensitization occurs. We report here two sets of experiments: (1) Rats were infused with dizocilpine through a subcutaneous mini-pump (0.1 mg/kg/day) during the induction of cocaine sensitization and, after 2 or 7 days of wash-out, were challenged with: cocaine, dizocilpine plus cocaine or 3-((+/-)2-carboxypiperazin-4-yl)-propyl-L-phosphonic acid (CPP) plus cocaine. Cocaine induced stereotypy scores significantly lower than that produced by the two drug combinations. Animals infused with dizocilpine alone did not present stereotypies when challenged either with dizocilpine or with dizocilpine plus cocaine. (2) Rats previously sensitized to cocaine received dizocilpine by infusion and daily cocaine treatments for a week. During the first days of infusion, sensitization appeared to be significantly decreased, but it resumed the initial intensity on days 6-7. After 2 and 9 days of wash-out, the expression of sensitization could be retrieved only by dizocilpine plus cocaine. Two distinct forms of sensitization to cocaine thus seem to exist: one dependent on and the second independent of NMDA receptor activity.

Further insights into the anti-aggregating activity of NMDA in human platelets.

1. In the present study the effect of N-methyl-D-aspartate (NMDA) on thromboxane B2 synthesis and on [Ca2+]i was studied in human platelets. 2. NMDA (10(-7) M) completely inhibited the synthesis of thromboxane B2 from exogenous arachidonic acid (AA), while it did not interfere with the aggregating effect of the thromboxane A2 receptor agonist U-46619. 3. NMDA (0.1 microM - 10 microM) dose-dependently increased intracellular calcium in washed platelets preloaded with fura 2 AM, and this effect was not additive with that of AA. 4. NMDA shifted the dose-response curve of AA to the right. At the highest AA concentrations platelet aggregation was not inhibited. 5. The antiaggregating effect of NMDA was not antagonized by N(G)-monomethyl-L-arginine (L-NMMA), a nitric oxide synthase (NOS) inhibitor. 6. Finally, NMDA (0.01 nM - 100 nM) associated with either aspirin or indomethacin significantly potentiated the antiaggregating activity of both cyclo-oxygenase inhibitors. 7. It was concluded that NMDA is a potent inhibitor of platelet aggregation and thromboxane B2 synthesis in human platelet rich plasma (PRP).