miR-23b/SP1/c-myc forms a feed-forward loop supporting multiple myeloma cell growth.

Deregulated microRNA (miR)/transcription factor (TF)-based networks represent a hallmark of cancer. We report here a novel c-Myc/miR-23b/Sp1 feed-forward loop with a critical role in multiple myeloma (MM) and Waldenstrom's macroglobulinemia (WM) cell growth and survival. We have found miR-23b to be downregulated in MM and WM cells especially in the presence of components of the tumor bone marrow milieu. Promoter methylation is one mechanism of miR-23b suppression in myeloma. In gain-of-function studies using miR-23b mimics-transfected or in miR-23b-stably expressing MM and WM cell lines, we observed a significant decrease in cell proliferation and survival, along with induction of caspase-3/7 activity over time, thus supporting a tumor suppressor role for miR-23b. At the molecular level, miR-23b targeted Sp1 3'UTR and significantly reduced Sp1-driven nuclear factor-κB activity. Finally, c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, transcriptionally repressed miR-23b. Thus MYC-dependent miR-23b repression in myeloma cells may promote activation of oncogenic Sp1-mediated signaling, representing the first feed-forward loop with critical growth and survival role in myeloma.

Mitochondrial DNA variability modulates mRNA and intra-mitochondrial protein levels of HSP60 and HSP75: experimental evidence from cybrid lines.

To explore possible relationships between mitochondrial DNA (mtDNA) polymorphism and the expression levels of stress-responder nuclear genes we assembled five cybrid cell lines by repopulating 143B.TK(-) cells, depleted of their own mtDNA (Rho(0) cells), with foreign mitochondria with different mtDNA sequences (lines H, J, T, U, X). We evaluated, at both basal and under heat stress conditions, gene expression (mRNA) and intra-mitochondrial protein levels of HSP60 and HSP75, two key components in cellular stress response. At basal conditions, the levels of HSP60 and HSP75 mRNA were lower in one cybrid (H) than in the others (p = 0.005 and p = 0.001, respectively). Under stress conditions, the H line over-expressed both genes, so that the inter-cybrid difference was abolished. Moreover, the HSP60 intra-mitochondrial protein levels differed among the cybrid lines (p = 0.001), with levels higher in H than in the other cybrid lines. On the whole, our results provide further experimental evidence that mtDNA variability influences the cell response to stressful conditions by modulating components involved in this response. Sentence summary of the article: the results reported in the present study provide important experimental evidence that in human cells mtDNA variability is able to influence the cellular response to heat stress by modulating both the transcription of genes involved in this response and their intra-mitochondrial protein levels.

Anti-inflammatory activity of linalool and linalyl acetate constituents of essential oils.

Linalool and linalyl acetate are the principal components of many essential oils known to possess several biological activities, attributable to these monoterpene compounds. In this work, we evaluated individually the anti-inflammatory properties of (-) linalool, that is, the natural occurring enantiomer, and its racemate form, present in various amounts in distilled or extracted essential oils. Because in the linalool-containing essential oils, linalyl acetate, is frequently present, we also examined the anti-inflammatory action of this monoterpene ester. Carrageenin-induced edema in rats was used as a model of inflammation. The experimental data indicate that both the pure enantiomer and its racemate induced, after systemic administration, a reduction of edema. Moreover, the pure enantiomer, at a dose of 25 mg/kg, elicited a delayed and more prolonged effect, while the racemate form induced a significant reduction of the edema only one hour after carrageenin administration. At higher doses, no differences were observed between the (-) enantiomer and the racemate; a further increase in the dose of both forms did not result in an increased effect at any time of observation. The effects of equi-molar doses of linalyl acetate on local edema were less relevant and more delayed than that of the corresponding alcohol. These finding suggest a typical pro-drug behavior of linalyl acetate. The results obtained indicate that linalool and the corresponding acetate play a major role in the anti-inflammatory activity displayed by the essential oils containing them, and provide further evidence suggesting that linalool and linalyl acetate-producing species are potentially anti-inflammatory agents.

Carbamazepine prevents imipramine-induced behavioural sensitization to the dopamine D(2)-like receptor agonist quinpirole.

Chronic treatment with antidepressants potentiates the behavioural sensitivity to the administration of dopamine receptor agonists. Such supersensitivity might be involved in the mechanism of action of antidepressant drugs, but it has also been suggested to play a role in the mechanisms underlying antidepressant treatment-related mania (i.e. antidepressant-induced mood switch and rapid cycling). Consistently to this hypothesis, we have recently shown that lithium salts, which are poorly effective in antidepressant-related mania, fail to prevent the development of imipramine-induced supersensitivity to the locomotor effect of the dopamine D(2)-like receptor agonist quinpirole. In the present paper, we report the ability of carbamazepine, an anticonvulsant with antimanic and mood stabiliser properties, to prevent the development of supersensitivity to the locomotor response to quinpirole induced by chronic treatment with imipramine. The present results, together with the results of our previous study, might contribute to explain the different responsiveness to lithium and carbamazepine observed in some manic patients, and are consistent with the clinical data suggesting that carbamazepine might be more effective than lithium in antidepressant-related mania.

The role of dopamine in the mechanism of action of antidepressant drugs.

The present paper reviews evidence on the effect of antidepressant treatments on dopamine transmission. Chronic treatment with antidepressant drugs potentiates the behavioural stimulant responses elicited by the stimulation of dopamine receptors, including reward-related behaviours. Moreover, antidepressants affect dopamine release in several brain areas. The reviewed literature is discussed in terms of the possible mechanisms underlying antidepressant-induced supersensitivity to dopamine-mediated behavioural responses, and of the possible implications for the therapeutic effect of these drugs. It is concluded that the potentiation of dopaminergic neurotransmission induced by chronic antidepressant treatments might contribute to their therapeutic effect.

Exploratory behaviour and grooming after repeated restraint and chronic mild stress: effect of desipramine.

In a previous study, we have recently shown that chronic treatment with desipramine either reduced or potentiated the locomotor response to the dopamine D(2)-like receptor agonist quinpirole, a behavioural response mediated by the mesolimbic dopamine system, depending on whether the animals were subjected, respectively, to repeated restraint or to chronic mild stress (different stressors randomly presented). In this study, we examined the interaction between prolonged exposure to either repeated restraint stress or chronic mild stress with the chronic administration of the antidepressant desipramine on two spontaneous behaviours, in which an involvement of the mesolimbic dopamine system has been suggested: novelty-induced exploratory activity and grooming. Exploratory activity in the open field was reduced by chronic mild stress regardless of the drug treatment, while it was not influenced by restraint stress. Desipramine reduced exploratory activity in rats subjected to restraint stress. Restraint stress increased grooming and desipramine reversed this effect, while increasing grooming in the chronic mild stress group. These findings suggest that antidepressants exert their effect by opposing the modifications induced by stress. The available experimental evidence is consistent with the hypothesis that an important role in the observed behavioural changes is played by the mesolimbic dopamine system.

Chronic lithium chloride fails to prevent imipramine-induced sensitization to the dopamine D(2)-like receptor agonist quinpirole.

Lithium salts, an effective antimanic treatment, are able to prevent the development of the dopaminergic behavioural supersensitivity induced by chronic treatment with neuroleptics, by denervation of the dopaminergic terminal fields and by rapid eye movements (REM) sleep deprivation, which is considered a model of mania. We have studied the effect of a lithium (LiCl) diet, inducing a lithium serum level in the range of therapeutic efficacy, on the development of the supersensitivity to the locomotor effect of the dopamine D(2)-like receptor agonist, quinpirole, induced by chronic treatment with the antidepressant drug, imipramine. The results show that lithium is not able to prevent the development of such behavioural supersensitivity. The present data suggest that antidepressant-induced dopaminergic supersensitivity might provide a useful model of those manic states induced by (or subsequent to) antidepressant treatments. Moreover, the finding is consistent with the view that antidepressant-induced dopaminergic supersensitivity might play a role in the therapeutic effect of these drugs (which is known to be augmented by lithium, and not antagonised). Finally, the results show that the dopaminergic supersensitivity induced by imipramine is qualitatively different from that induced by neuroleptics or denervation of the dopaminergic terminal fields.

Different effect of desipramine on locomotor activity in quinpirole-treated rats after repeated restraint and chronic mild stress.

We have studied the effect of chronic treatment with the tricyclic antidepressant drug desipramine on locomotor activity in rats challenged with the administration of the D2-like dopamine agonist quinpirole, after prolonged exposure to two different stress regimens, repeated restraint stress and chronic mild stress (different stressors randomly presented). These stress schedules have been previously reported to influence in opposite ways the sensitivity to the locomotor response mediated by the stimulation of mesolimbic dopamine receptors. In particular, repeated restraint has been reported to induce an increased response to the locomotor effect of amphetamine, while chronic mild stress has been reported to induce a decreased locomotor response to quinpirole. In the present study, repeated restraint stress failed to influence the locomotor activity after challenge with quinpirole, while chronic mild stress reduced this response. Chronic treatment with desipramine failed to influence this response in the control group, but exerted opposite effects in the two stressed groups. In particular, chronic desipramine reduced locomotor activity in quinpirole-treated rats in the restraint stress group, and increased it in the chronic mild stress group, thus preventing the subsensitivity induced by this stress regimen. The present results, taken together with results from earlier studies, are consistent with the hypothesis that the effect of antidepressants on the sensitivity of the mesolimbic dopamine receptors mediating the locomotor behavioural response tends to be opposite with respect to that exerted by stress, regardless of its direction. However, since we failed to show an increased locomotor activity after quinpirole challenge in the repeated restraint group, this hypothesis remains to be demonstrated. The two stress schedules reduced body weight gain in a similar way, therefore their different effects do not seem to be due to a difference in stress severity. Thus, the observation that both stress schedules reduced body weight gain in a similar way, but only chronic mild stress reduced the sensitivity to the locomotor response to quinpirole, shows that this effect is not an artefact of body weight decrease.

Diurnal variation in the effect of chronic mild stress on sucrose intake and preference.

Diurnal variation (depression worse in the morning) is one of the diagnostic criteria for the melancholic subtype of major depression. This study examined diurnal variation in the effects of chronic mild stress (CMS), an animal model of depression, by testing Wistar rats at different phases of the light-dark cycle. CMS decreased sucrose intake and sucrose preference in animals tested at the start of the dark phase (the most active period in this nocturnal species), but not in animals tested during the light phase. CMS also decreased body weight in both groups; however, the effects of CMS on sucrose intake in the dark phase were not secondary to body weight changes. On the contrary, loss of body weight led to underestimates in the magnitude of the effects of CMS on sucrose intake. The results support the validity of the CMS procedure as a model of melancholia. The discussion addresses criticisms of this position that have been raised in two recent publications.

Dizocilpine prevents the enhanced locomotor response to quinpirole induced by repeated electroconvulsive shock.

Repeated administration of electroconvulsive shock, as expected, potentiated the locomotor stimulant response to quinpirole (0.3 mg/kg s.c.), a dopamine D2-like receptor agonist. Chronic, but not acute, treatment with the NMDA receptor non-competitive antagonist dizocilpine (0.3 mg/kg i.p.) prevented electroconvulsive shock-induced potentiation of quinpirole locomotor response. These results suggest that NMDA receptor activation is necessary for the development of supersensitivity to dopamine receptor agonists produced by repeated electroconvulsive shock. The relevance of this observation in regard to the mechanism of electroconvulsive shock therapeutic effect is discussed.

Role of D1 and alpha1 receptors in the enhanced locomotor response to dopamine D2-like receptor stimulation induced by repeated electroconvulsive shock.

We previously reported that, in rats chronically treated with the antidepressant drug imipramine, the enhanced locomotor response to the D2-like receptor agonist quinpirole became less sensitive to the inhibitory effect of the D1 receptor antagonist SCH 23390 and more sensitive to the inhibitory effect of the alpha1 receptor antagonist prazosin. In this study, we show that in electroconvulsive shock-treated rats these antagonists behave in the opposite manner to that observed in imipramine-treated rats, with SCH 23390 being highly effective and prazosin ineffective in antagonizing the locomotor response to quinpirole. The possibility that these differences may reflect some of the clinical characteristics of these antidepressant treatments is discussed.

Social influences on morphine conditioned place preference.

Social defeat in dominant male rats has previously been shown to result in a loss of rank, together with decreased consumption of a palatable sucrose solution, suggestive of a decrease in hedonic tone. The present study further investigated the effect of defeat on hedonic capacity using the conditioned place preference paradigm. Stable dominance hierarchies were determined in pairs of male PVG rats by repeated observation of agonistic behaviour at the onset of the dark phase of the dark-light cycle. Dominant animals and singly housed animals were then subjected to defeat by a male of the aggressive Tryon Maze Dull (TMD) strain, either 1 (singly housed animals only) 3 or 7 days prior to the first conditioning session. Each animal was tested for place conditioning with morphine (1 mg/kg i.p.) using one rewarded and one non-rewarded conditioning trial. In this paradigm, dominant animals as well as singly housed animals showed an increase in time spent in the drug-associated side but their submissive partners did not. Three days following the defeat of a dominant animal by a TMD, place conditioning to morphine was absent in the defeated animals, but was now present in their submissive partners. Seven days following defeat, conditioning was absent in defeated animals that were now submissive, but present in defeated animals that had maintained their dominant status. In isolated animals, place conditioning was absent 1 day following defeat, but was present 3 and 7 days post-defeat. These findings suggest that morphine-induced place conditioning is influenced by both the experience of defeat and the effects of defeat on social status.

Anti-anhedonic actions of the novel serotonergic agent flibanserin, a potential rapidly-acting antidepressant.

Chronic exposure to mild unpredictable stress has previously been found to depress the consumption of palatable sweet solutions and to block the formation of conditioned place preferences; these effects are reversed by chronic treatment with tricyclic or atypical antidepressant drugs. The present study was designed to evaluate the antidepressant-like activity in this model of flibaserin (BIMT-17), a novel serotonergic agent with 5-HT1A receptor agonist and 5-HT2 receptor antagonist properties. Two experiments were conducted, using rats (experiment 1) and mice (experiment 2). In experiment 1, decreases in sucrose intake were seen in rats exposed to chronic mild stress, but the effect was unreliable in this study, and sucrose testing was terminated after 7 weeks of stress. Beginning after 5 weeks of stress, groups of control and stressed animals were treated daily with vehicle, fluoxetine (5 mg/kg) or flibanserin (5, 10 or 20 mg/kg). After 6 weeks of treatment, all animals were tested for acquisition of food-reinforced place preference conditioning. Conditioning was seen in all groups other than the vehicle-treated stressed animals. We also tested the locomotor stimulant effect of a single injection of the dopamine D2/D3 receptor agonist quinpirole (0.2 mg/kg). The effect of quinpirole was potentiated by fluoxetine in control animals, and by both fluoxetine and flibanserin (all doses) in stressed animals. In experiment 2, long-lasting decreases in sucrose intake were seen in mice exposed to chronic mild stress. The effects were reversed by chronic (4 weeks) treatment with fluoxetine (5 mg/kg) or flibanserin (2.5 or 5 mg/kg); the full effect of flibanserin was seen after the first injection. All animals received a single injection of raclopride (0.1 mg/kg) immediately prior to a sucrose intake test on day 27 of drug treatment. Raclopride decreased sucrose intake only in the three drug-treated stressed groups. The results support a rapid antidepressant-like action of flibanserin, and suggest that this effect involves sensitization of dopamine D2/D3 receptor-mediated transmission.

Attenuation of sucrose consumption in mice by chronic mild stress and its restoration by imipramine.

Chronic exposure to mild unpredictable stressors (CMS) has previously been found to reduce the consumption of palatable, sweet solutions in rats. In the present study, the utility of this procedure was assessed in mice. Male AP mice subjected to CMS showed reduced consumption of a 2% or 4% sucrose solution. This effect was reversed by chronic (3 weeks) treatment with the tricyclic antidepressant imipramine (20 mg/kg per day). These results extend previous reports of a generalized decrease in sensitivity to reward (anhedonia) in rats caused by CMS and the efficacy of antidepressant treatment in this paradigm. Chronic unpredictable mild stress in mice appears to provide a realistic animal model of depression.

Loss of social status: preliminary evaluation of a novel animal model of depression.

Stable dominance hierarchies were determined in pairs of male Lister hooded rats, by repeated observation of agonistic behaviour at the onset of the dark phase of the light-dark cycle. No lasting alterations in dominance behaviour were caused by subjecting the dominant member of the pair either to restraint stress or to defeat by another dominant animal. However, defeat of the dominant animal by a male of the aggressive Tryon Maze Dull (TMD) strain caused a loss of dominant status in the home cage which lasted at least 7 days. Repeated weekly defeat by TMD animals decreased both home cage dominance behaviour and consumption of a palatable sucrose solution, relative to non-defeated animals; both behaviours were normalized in defeated animals by 3 weeks of treatment with the tricyclic antidepressant imipramine.

Effects of chronic mild stress on performance in behavioural tests relevant to anxiety and depression.

Chronic exposure to mild unpredictable stress (CMS) has previously been found to depress the consumption of a weak (1%) sucrose solution by rats. This effect was confirmed in each of three experiments in the present study, following which behaviour was examined in other tests relevant to either depression or anxiety. CMS did not significantly affect behaviour in the social interaction test and caused an anxiolytic-like profile in the elevated plus-maze. CMS increased submissive behaviour in the resident-intruder test, and decreased male sexual behaviour. The latter effect was more pronounced in animals reared in isolation from the time of weaning; isolation rearing did not influence sexual behaviour in nonstressed animals. Isolation rearing also potentiated the effect of CMS on sucrose drinking, in both male and female rats. These results support the relevance of the CMS procedure as a potential animal model of depression.

Antidepressant-like effect of selective dopamine D1 receptor agonists in the behavioural despair animal model of depression.

We compared the effect of two selective dopamine D1 receptor agonists, SKF 38393 ((+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol.HCl) and A68930 ((1R,3S)-1-aminomethyl-5,6-dihydroxy-3-phenylisochroman.HCl), and that of imipramine in the behavioural despair model of depression. The dopamine D1 receptor agonists and imipramine showed an anti-immobility effect. Moreover we found that the 'antidepressant' effect of imipramine in the behavioural despair test was antagonized by SCH 23390, a selective dopamine D1 receptor blocker. The results further support the hypothesis that dopamine D1 receptor stimulation plays an important role in the mechanism of action of antidepressants and suggest that dopamine D1 receptor agonists might be considered as potential antidepressant drugs.

The NMDA receptor antagonist MK-801 prevents imipramine-induced supersensitivity to quinpirole.

Chronic treatment with imipramine enhanced the locomotor stimulant response to quinpirole (0.3 mg/kg s.c.), a dopamine D2/D3 receptor agonist. Chronic, but not acute, blockade of the NMDA receptor with the non-competitive antagonist MK-801 (0.3 mg/kg i.p.) prevented the imipramine-induced potentiation of the quinpirole effect. The results suggest that NMDA receptors play a role in the development of supersensitivity to dopamine receptor agonists produced by chronic antidepressant treatment.

TRH activates mesolimbic dopamine system: behavioural evidence.

The effect of TRH pretreatment on locomotor activity and stereotypy induced by d-amphetamine was examined in rats. The results show that TRH potentiates d-amphetamine-induced locomotor activity, but not d-amphetamine-induced stereotypy, suggesting that this neuropeptide selectively activates the mesolimbic dopamine system.