Bmcc1s interacts with the phosphate-activated glutaminase in the brain.

Bmcc1s, a brain-enriched short isoform of the BCH-domain containing molecule Bmcc1, has recently been shown to interact with the microtubule-associated protein MAP6 and to regulate cell morphology. Here we identified kidney-type glutaminase (KGA), the mitochondrial enzyme responsible for the conversion of glutamine to glutamate in neurons, as a novel partner of Bmcc1s. Co-immunoprecipitation experiments confirmed that Bmcc1s and KGA form a physiological complex in the brain, whereas binding and modeling studies showed that they interact with each other. Overexpression of Bmcc1s in mouse primary cortical neurons impaired proper mitochondrial targeting of KGA leading to its accumulation within the cytoplasm. Thus, Bmcc1s may control the trafficking of KGA to the mitochondria.

Alterations of CXCR4 function in µ-opioid receptor-deficient glia.

The chemokine receptor CXCR4 and the µ-opioid receptor (MOR) are G-protein-coupled receptors that are essential for normal function of the nervous and immune systems. Several studies have suggested that MOR is a key regulator of CXCR4 in the brain; however, the molecular basis of the opioid-chemokine interaction is not fully understood, and it may involve different mechanisms in neuronal and glial cells. Our previous studies demonstrated that MOR stimulation specifically upregulates the protein ferritin heavy chain - an inhibitor of CXCR4 - in neurons, and suggested that additional mechanisms could be operative in glia. In this study, we investigated CXCR4 function in brains and astroglial cultures deprived of MOR. Reduced coupling of CXCR4 to G-proteins was found in brain slices and tissue homogenates of MOR(-/-) mice as compared with wild-type controls. CXCR4-induced signaling was also reduced in glial cultures from MOR(-/-) mice, as shown by analysis of CXCR4 downstream targets (Akt and ERK1/2). Pharmacological studies with δ-opioid receptor (DOR)-specific ligands suggested that DOR-CXCR4 interactions are implicated in the inhibition of CXCR4 in MOR-deficient cells both in vitro and in vivo. Moreover, increased CXCR4/DOR co-immunoprecipitation was found in brain tissue and cultured glia from MOR(-/-) mice. Importantly, CXCR4 function was restored by pretreatment with a DOR antagonist. Overall, these findings indicate that DOR plays a crucial role in the regulation of CXCR4 in glia, probably via silent receptor heterodimers. The data also suggest that the opiate system interferes with normal CXCR4 function in different ways, depending on receptor subtypes.

Disruption of neuronal CXCR4 function by opioids: preliminary evidence of ferritin heavy chain as a potential etiological agent in neuroAIDS.

The chemokine CXCL12 and its receptor, CXCR4, regulate neuronal migration, differentiation, and survival. Alterations of CXCL12/CXCR4 signaling are implicated in different neuropathologies, including the neurological complications of HIV infection. Opiates are important co-factors for progression to neuroAIDS and can disrupt the CXCL12/CXCR4 axis in vitro and in vivo. This paper will review recently identified mechanisms of opiate-induced CXCR4 impairment in neurons and introduce results from pilot studies in human brain tissue, which highlight the role of the protein ferritin heavy chain in HIV neuropathology in patients with history of drug abuse.

Gamma-hydroxybutyrate does not maintain self-administration but induces conditioned place preference when injected in the ventral tegmental area.

Gamma-hydroxybutyric acid (GHB) is an endogenous brain substance that has diverse neuropharmacological actions, including rewarding properties in different animal species and in humans. As other drugs of abuse, GHB affects the firing of ventral tegmental neurons (VTA) in anaesthetized animals and hyperpolarizes dopaminergic neurons in VTA slices. However, no direct behavioural data on the effects of GHB applied in the VTA or in the target regions of its dopaminergic neurons, e.g. the nucleus accumbens (NAc), are available. Here, we investigated the effects of various doses of intravenous GHB in maintaining self-administration (from 0.001 to 10 mg/kg per infusion), and its ability to induce conditioned place preference (CPP) in rats when given orally (175-350 mg/kg) or injected directly either in the VTA or NAc (from 10 to 300 microg/0.5 microl per side). Our results indicate that while only 0.01 mg/kg per infusion GHB maintained self-administration, although not on every test day, 350 mg/kg GHB given orally induced CPP. CPP was also observed when GHB was injected in the VTA (30-100 microg/0.5 microl per side) but not in the NAc. Together with recent in-vitro findings, these results suggest that the rewarding properties of GHB mainly occur via disinhibition of VTA dopaminergic neurons.

Morphine increases brain levels of ferritin heavy chain leading to inhibition of CXCR4-mediated survival signaling in neurons.

This study focuses on the effect of mu-opioid receptor agonists on CXCR4 signaling in neurons and the mechanisms involved in regulation of neuronal CXCR4 by opiates. The data show that CXCR4 is negatively modulated by long-term morphine treatments both in vitro and in vivo; CXCR4 inhibition is caused by direct stimulation of mu-opioid receptors in neurons, leading to alterations of ligand-induced CXCR4 phosphorylation and upregulation of protein ferritin heavy chain (FHC), a negative intracellular regulator of CXCR4. Reduced coupling of CXCR4 to G-proteins was found in the brain of morphine-treated rats, primarily cortex and hippocampus. CXCR4-induced G alpha(i)/G betagamma activities were suppressed after 24 h treatment of cortical neurons with morphine or the selective mu-opioid agonist DAMGO (D-Ala2-N-Me-Phe(4)-glycol(5)-enkephalin), as shown by analysis of downstream targets of CXCR4 (i.e., cAMP, Akt, and ERK1/2). These agonists also prevented CXCL12-induced phosphorylation of CXCR4, indicating a deficit of CXCR4 activation in these conditions. Indeed, morphine (or DAMGO) inhibited prosurvival signaling in neurons. These effects are not attributable to a reduction in CXCR4 expression or surface levels but rather to upregulation of FHC by opioids. The crucial role of FHC in inhibition of neuronal CXCR4 was confirmed by in vitro and in vivo RNA interference studies. Overall, these findings suggest that opiates interfere with normal CXCR4 function in the brain. By this mechanism, opiates could reduce the neuroprotective functions of CXCR4 and exacerbate neuropathology in opiate abusers who are affected by neuroinflammatory/infectious disorders, including neuroAIDS.

GTPgammaS incorporation in the rat brain: a study on mu-opioid receptors and CXCR4.

Chemokine and opioid receptors are G-protein-coupled receptors that play important roles in both the central nervous system and the immune system. The long-term goal of our research is to establish whether opioids regulate the activity of the chemokine receptor CXCR4 (one of the major HIV co-receptors) in the brain. In this research, we studied the anatomical distribution of functional receptors in young and adult animals by using the [(35)S]GTPgammaS "binding" assay as an indication of G-protein activation by CXCL12 (the natural CXCR4 ligand) or by mu-opioid agonists. Brain slices or homogenates from Holtzmann rats of different ages (from 2 to 21 days old and adult animals) were treated with CXCL12 (0.001-100 nM), D: -ala2,MePhe4,gly-ol5]enkephalin (DAMGO; 0.0003-10 microM) or morphine (0.0003-10 microM) and then processed for the assay. Our results show stimulation of both mu-OR and CXCR4 in several brain areas, including cortex and hippocampus (p < 0.001); this effect is dose and age dependent, and the magnitude of response varies among different brain regions. Furthermore, AMD3100 (100 ng/ml), a specific CXCR4 antagonist, abolished CXCL12 stimulation in all the brain regions analyzed (p < 0.001). Our findings suggest a similar pattern of expression for mu-OR and CXCR4 in the brain, supporting the possibility of an interaction between the two G-protein-coupled receptors in vivo. This might be relevant to the role of opiates in HIV neuropathogenesis.

Effects of naltrexone on cocaine- and sucrose-seeking behaviour in response to associated stimuli in rats.

The non-selective opioid receptor antagonist naltrexone reduces cocaine-induced reinstatement of drug-seeking behaviour in abstinent rats. The current study sought to determine whether the opioid system is also involved in cocaine-seeking behaviour induced by cocaine-associated stimuli in abstinent rats. Adult male rats were trained to press a lever either to self-administer cocaine or to obtain sucrose pellets in the presence of distinctive discriminative and conditioned stimuli. After a period of extinction, re-exposure to cocaine-associated cues selectively elicited robust and enduring responding at the active lever; sucrose pellet-associated cues revived seeking behaviour less pronouncedly. Pretreatment with naltrexone (0.25, 1, 2.5 mg/kg s.c., 20 min before reinstatement tests) dose dependently prevented cue-induced cocaine-seeking behaviour, whereas (2.5 mg/kg s.c.) did not affect the degree of cue-induced sucrose-seeking behaviour. These results provide the first evidence that naltrexone influences cocaine seeking induced by conditioned stimuli in abstinent rats; this effect appears selective for cocaine reinstatement as opposed to a non-drug reinforcer.

Stimulation of serotonin2C receptors influences cocaine-seeking behavior in response to drug-associated stimuli in rats.

RATIONALE: It has been suggested that the increase in serotonin transmission induced by indirect agonists such as fenfluramine and fluoxetine attenuates cue-elicited reinstatement of cocaine-seeking in rats through a 5-HT2C receptor-dependent mechanism. OBJECTIVE: We investigated whether Ro 60-0175, a nonselective 5-HT2B-2C agonist, influences cue-elicited reinstatement of cocaine-seeking behavior. We evaluated the 5-HT2C receptor's role in Ro 60-0175 by studying its interaction with SB-242,084, a selective 5-HT2C antagonist. The study also explored whether Ro 60-0175 influences cue-elicited seeking behavior associated with sucrose, a highly palatable nutritive reinforcer. MATERIALS AND METHODS: Different groups of free-feeding rats were trained to associate discriminative stimuli (SDs) with the availability of cocaine or a sucrose pellet or no-reward in two-lever operant cages. Cocaine and sucrose pellets were available under an FR1 schedule of reinforcement, and each reinforcer was followed by a 20-s timeout signaled by a cue light coming above the active lever. After extinction of reinforced responding in the absence of cue, the reinforcer-associated stimuli were reintroduced in reinstatement sessions in which reinforcers were withheld. RESULTS: Ro 60-0175, at IP doses from 0.1 to 1 mg/kg, dose-dependently reduced cocaine-seeking behavior, while 1 mg/kg had no such effect for the sucrose pellet. Pretreatment with 1 mg/kg SC SB-242,084 completely prevented the effect on cocaine-seeking behavior. CONCLUSIONS: These findings, provided they can be extrapolated to abstinent human addicts, suggest therapeutic potential for the selective 5-HT2C agonist in preventing cue-controlled cocaine-seeking and relapse.

Genotype-dependent activity of tryptophan hydroxylase-2 determines the response to citalopram in a mouse model of depression.

Polymorphism of tryptophan hydroxylase, the rate-limiting enzyme in the synthesis of brain serotonin (5-HT), is associated with less synthesis of brain 5-HT in DBA/2J and BALB/c than in C57BL/6J and 129/Sv mice. We selected the forced swimming test, a mouse model used to assess the antidepressant potential of drugs, and neurochemical techniques to study strain differences in the response to citalopram, a selective 5-HT reuptake inhibitor. Citalopram reduced immobility time in C57BL/6J and 129/Sv mice but had no such effect in DBA/2J and BALB/c mice. The drug reduced accumulation of 5-hydroxytryptophan (5-HTP), an indicator of 5-HT synthesis, in C57BL/6J and 129/Sv mice but much less in DBA/2J and BALB/c mice. Pretreatment with tryptophan raised 5-HTP accumulation and reinstated the antidepressant-like effect of citalopram in DBA/2J and BALB/c mice, whereas pharmacological inhibition of 5-HT synthesis prevented the effect of citalopram in C57BL/6J and 129/Sv mice. Because there were no strain differences in catecholamine synthesis, locomotor activity, and brain levels of citalopram at the end of the behavioral test, the results suggest that the failure of citalopram to reduce immobility time in DBA/2J and BALB/c mice is attributable to genotype-dependent impairment of 5-HT synthesis. Interstrain comparisons could probably be a useful strategy for understanding the mechanisms underlying the response to selective serotonin reuptake inhibitors.

Potential antidepressant properties of IDN 5491 (hyperforin-trimethoxybenzoate), a semisynthetic ester of hyperforin.

Hyperforin is one of the possible active principles mediating the antidepressant activity of Hypericum perforatum L. extracts. The ester derivative IDN 5491 (hyperforin-trimethoxybenzoate) showed antidepressant-like properties in the forced swimming test (FST) in rats, with no effect on open-field activity, when given as three intraperitoneal injections in 24 h at 3.125 and 6.25 mg/kg. The plasma concentrations of IDN 5491 were 30-50 microM, and those of hyperforin much lower but still close to those after effective doses of hyperforin-dicyclohexylammonium and Hypericum extract. This suggests that hyperforin plays a role in the antidepressant-like effect of the ester and of Hypericum extract. In vitro binding and uptake data showed that IDN 5491 is inactive on a wide panel of CNS targets at a concentration (14 microM) much higher than that measured in the brain of treated rats (0.3 microM). Like the extract, the antidepressant-like effect of IDN 5491 was blocked by (-)-sulpiride, a selective D2 receptor antagonist and by BD-1047, a selective sigma1 antagonist. Ex-vivo binding studies showed that brain sigma1 receptors are occupied after in vivo treatment with IDN 5491, possibly by an unknown metabolite or by endogenous ligand induced by hyperforin.

Selective antagonist at D3 receptors, but not non-selective partial agonists, influences the expression of cocaine-induced conditioned place preference in free-feeding rats.

The non-selective dopamine (DA) D(3) partial agonist BP 897 influenced rats' seeking behavior induced by cocaine-associated cues but there are contradictions about its ability to modulate cocaine-induced conditioned place preference (CPP), and mechanisms involved. We therefore re-evaluated its activity on both acquisition and expression of these behaviors, taking into consideration the actual brain concentrations of unchanged drug and its potential active metabolite 1-(2-methoxyphenyl)-piperazine (oOCH(3)PP), as well as its negative motivational properties. BP 897 induced conditioned place aversion (CPA) at 3 mg/kg, but not at 0.3 and 1 mg/kg. However, in this range of amply spaced doses BP 897 did not affect the acquisition and expression of cocaine (10 mg/kg i.p.) CPP in rats, although its brain concentrations were well above those affecting in vitro D(3) receptors. Concentrations of oOCH(3)PP were below the limits of quantification of the analytical procedure. As concerns the expression behavior, its structurally and pharmacologically related derivative N-[4-[4-(2-methoxyphenyl)piperazin-1-yl]butyl]benzo[b]furan-2-carboxamide (1 and 3 mg/kg, i.p.) also had no such effect. By contrast, the selective D(3) receptor antagonist SB-277011-A (3 mg/kg, i.p.) antagonized the expression of cocaine-induced CPP, supporting the suggestion that "full" antagonist activity at D(3) receptors is necessary to prevent 10 mg/kg cocaine-induced place conditioning in free-feeding rats.

A single high dose of cocaine induces behavioural sensitization and modifies mRNA encoding GluR1 and GAP-43 in rats.

Neuroadaptive changes underlying repeated exposure to cocaine-induced behavioural sensitization have been related to modification in the pattern of synaptic connectivity and excitatory transmission. Remarkably, even a single exposure to abused drugs is sufficient to elicit lasting behavioural sensitization. The present study investigated whether in Sprague-Dawley rats a single, behavioural sensitizing dose of cocaine is sufficient to induce changes in the mRNA levels of growth-associated protein 43 (GAP-43), an important protein in mediating experience-dependent plasticity and synaptic reorganization, and of glutamate receptor 1 (GluR1), a subunit of AMPA glutamate receptors, a protein that is up-regulated with repeated cocaine. Single exposure to 20, but not 10 mg/kg cocaine induced locomotor sensitization to a second injection of 10 mg/kg cocaine, observed at 24 h, 48 h and 7 days. Single dose of 20 but not 10 mg/kg cocaine 48 h before scheduled death significantly enhanced GluR1 and GAP-43 mRNA expression in the nucleus accumbens (NAc), both shell and core subregions, and ventral tegmental area (VTA). No changes were found in the levels of mRNA for GluR1 and GAP-43 in the frontal cortex, caudate putamen, dentate gyrus of hippocampus and basolateral nucleus of the amygdala after the single dose of 20 mg/kg cocaine. These results further strengthen the involvement of NAc and VTA in the behavioural sensitization and suggest a role of GAP-43 in the synaptic reorganization associated to drug abuse.