Prazosin and cyproheptadine in combination in the treatment of alcohol use disorder: A randomized, double-blind, placebo-controlled trial.

BACKGROUND AND AIMS: Pre-clinical studies suggest that the simultaneous blockade of the α1b and 5HT2A receptors may be effective in reducing alcohol consumption. This study aimed to assess the efficacy and safety of prazosin (α1b blocker) and cyproheptadine (5HT2A blocker) combination in decreasing total alcohol consumption (TAC) in alcohol use disorder (AUD). DESIGN, SETTING AND PARTICIPANTS: This was a double-blind, parallel group, placebo-controlled, Phase 2, randomized clinical trial conducted in 32 addiction treatment centres in France. A total of 108 men and 46 women with severe AUD took part. INTERVENTION: Participants were randomly assigned to one of the following 3-month treatments: (1) low-dose group (LDG) receiving 8 mg cyproheptadine and 5 mg prazosin extended-release (ER) formulation daily; (2) high-dose group (HDG) receiving 12 mg cyproheptadine and 10 mg prazosin ER daily; and (3) placebo group (PG) receiving placebo of cyproheptadine and prazosin ER. A total of 154 patients were randomized: 54 in the PG, 54 in the LDG and 46 in the HDG. MEASUREMENTS: The primary outcome was TAC change from baseline to month 3. FINDINGS: A significant main treatment effect in the change in TAC was found in the intent-to-treat population (P = 0.039). The HDG and LDG showed a benefit in the change in TAC from baseline to month 3 compared with PG: -23.6 g/day, P = 0.016, Cohen's d = -0.44; -18.4 g/day, P = 0.048 (Bonferroni correction P < 0.025), Cohen's d = -0.36. In a subgroup of very high-risk drinking-level participants (> 100 g/day of pure alcohol for men and > 60 g/day for women), the difference between the HDG and the PG in the primary outcome was -29.8 g/day (P = 0.031, Cohen's d = -0.51). The high and low doses were well-tolerated with a similar safety profile. CONCLUSIONS: A randomized controlled trial of treatment of severe alcohol use disorder with a cyproheptadine-prazosin combination for 3 months reduced drinking by more than 23 g per day compared with placebo. A higher dose combination was associated with a larger magnitude of drinking reduction than a lower dose combination while showing similar safety profile.

NLRP6 controls pulmonary inflammation from cigarette smoke in a gut microbiota-dependent manner.

Chronic obstructive pulmonary disease (COPD) is a major health issue primarily caused by cigarette smoke (CS) and characterized by breathlessness and repeated airway inflammation. NLRP6 is a cytosolic innate receptor controlling intestinal inflammation and orchestrating the colonic host-microbial interface. However, its roles in the lungs remain largely unexplored. Using CS exposure models, our data show that airway inflammation is strongly impaired in Nlrp6-deficient mice with drastically fewer recruited neutrophils, a key cell subset in inflammation and COPD. We found that NLRP6 expression in lung epithelial cells is important to control airway and lung tissue inflammation in an inflammasome-dependent manner. Since gut-derived metabolites regulate NLRP6 inflammasome activation in intestinal epithelial cells, we investigated the link between NLRP6, CS-driven lung inflammation, and gut microbiota composition. We report that acute CS exposure alters gut microbiota in both wild-type (WT) and Nlrp6-deficient mice and that antibiotic treatment decreases CS-induced lung inflammation. In addition, gut microbiota transfer from dysbiotic Nlrp6-deficient mice to WT mice decreased airway lung inflammation in WT mice, highlighting an NLRP6-dependent gut-to-lung axis controlling pulmonary inflammation.

Aberrant survival of hippocampal Cajal-Retzius cells leads to memory deficits, gamma rhythmopathies and susceptibility to seizures in adult mice.

Cajal-Retzius cells (CRs) are transient neurons, disappearing almost completely in the postnatal neocortex by programmed cell death (PCD), with a percentage surviving up to adulthood in the hippocampus. Here, we evaluate CR's role in the establishment of adult neuronal and cognitive function using a mouse model preventing Bax-dependent PCD. CRs abnormal survival resulted in impairment of hippocampus-dependent memory, associated in vivo with attenuated theta oscillations and enhanced gamma activity in the dorsal CA1. At the cellular level, we observed transient changes in the number of NPY+ cells and altered CA1 pyramidal cell spine density. At the synaptic level, these changes translated into enhanced inhibitory currents in hippocampal pyramidal cells. Finally, adult mutants displayed an increased susceptibility to lethal tonic-clonic seizures in a kainate model of epilepsy. Our data reveal that aberrant survival of a small proportion of postnatal hippocampal CRs results in cognitive deficits and epilepsy-prone phenotypes in adulthood.

P2X7 Receptor Promotes Mouse Mammary Cancer Cell Invasiveness and Tumour Progression, and Is a Target for Anticancer Treatment.

The P2X7 receptor is an ATP-gated cation channel with a still ambiguous role in cancer progression, proposed to be either pro- or anti-cancerous, depending on the cancer or cell type in the tumour. Its role in mammary cancer progression is not yet defined. Here, we show that P2X7 receptor is functional in highly aggressive mammary cancer cells, and induces a change in cell morphology with fast F-actin reorganization and formation of filopodia, and promotes cancer cell invasiveness through both 2- and 3-dimensional extracellular matrices in vitro. Furthermore, P2X7 receptor sustains Cdc42 activity and the acquisition of a mesenchymal phenotype. In an immunocompetent mouse mammary cancer model, we reveal that the expression of P2X7 receptor in cancer cells, but not in the host mice, promotes tumour growth and metastasis development, which were reduced by treatment with specific P2X7 antagonists. Our results demonstrate that P2X7 receptor drives mammary tumour progression and represents a pertinent target for mammary cancer treatment.

Complementarity of gluCEST and 1 H-MRS for the study of mouse models of Huntington's disease.

Identification of relevant biomarkers is fundamental to understand biological processes of neurodegenerative diseases and to evaluate therapeutic efficacy. Atrophy of brain structures has been proposed as a biomarker, but it provides little information about biochemical events related to the disease. Here, we propose to identify early and relevant biomarkers by combining biological specificity provided by 1 H-MRS and high spatial resolution offered by gluCEST imaging. For this, two different genetic mouse models of Huntington's disease (HD)-the Ki140CAG model, characterized by a slow progression of the disease, and the R6/1 model, which mimics the juvenile form of HD-were used. Animals were scanned at 11.7 T using a protocol combining 1 H-MRS and gluCEST imaging. We measured a significant decrease in levels of N-acetyl-aspartate, a metabolite mainly located in the neuronal compartment, in HD animals, and the decrease seemed to be correlated with disease severity. In addition, variations of tNAA levels were correlated with striatal volumes in both models. Significant variations of glutamate levels were also observed in Ki140CAG but not in R6/1 mice. Thanks to its high resolution, gluCEST provided complementary insights, and we highlighted alterations in small brain regions such as the corpus callosum in Ki140CAG mice, whereas the glutamate level was unchanged in the whole brain of R6/1 mice. In this study, we showed that 1 H-MRS can provide key information about biological processes occurring in vivo but was limited by the spatial resolution. On the other hand, gluCEST may finely point to alterations in unexpected brain regions, but it can also be blind to disease processes when glutamate levels are preserved. This highlights in a practical context the complementarity of the two methods to study animal models of neurodegenerative diseases and to identify relevant biomarkers.

Protein kinase Cθ controls type 2 innate lymphoid cell and TH2 responses to house dust mite allergen.

BACKGROUND: Protein kinase C (PKC) θ, a serine/threonine kinase, is involved in TH2 cell activation and proliferation. Type 2 innate lymphoid cells (ILC2s) resemble TH2 cells and produce the TH2 cytokines IL-5 and IL-13 but lack antigen-specific receptors. The mechanism by which PKC-θ drives innate immune cells to instruct TH2 responses in patients with allergic lung inflammation remains unknown. OBJECTIVES: We hypothesized that PKC-θ contributes to ILC2 activation and might be necessary for ILC2s to instruct the TH2 response. METHODS: PRKCQ gene expression was assessed in innate lymphoid cell subsets purified from human PBMCs and mouse lung ILC2s. ILC2 activation and eosinophil recruitment, TH2-related cytokine and chemokine production, lung histopathology, interferon regulatory factor 4 (IRF4) mRNA expression, and nuclear factor of activated T cells (NFAT1) protein expression were determined. Adoptive transfer of ILC2s from wild-type mice was performed in wild-type and PKC-θ-deficient (PKC-θ-/-) mice. RESULTS: Here we report that PKC-θ is expressed in both human and mouse ILC2s. Mice lacking PKC-θ had reduced ILC2 numbers, TH2 cell numbers and activation, airway hyperresponsiveness, and expression of the transcription factors IRF4 and NFAT1. Importantly, adoptive transfer of ILC2s restored eosinophil influx and IL-4, IL-5 and IL-13 production in lung tissue, as well as TH2 cell activation. The pharmacologic PKC-θ inhibitor (Compound 20) administered during allergen challenge reduced ILC2 numbers and activation, as well as airway inflammation and IRF4 and NFAT1 expression. CONCLUSIONS: Therefore our findings identify PKC-θ as a critical factor for ILC2 activation that contributes to TH2 cell differentiation, which is associated with IRF4 and NFAT1 expression in allergic lung inflammation.

The Combination of Marketed Antagonists of α1b-Adrenergic and 5-HT2A Receptors Inhibits Behavioral Sensitization and Preference to Alcohol in Mice: A Promising Approach for the Treatment of Alcohol Dependence.

Alcohol-dependence is a chronic disease with a dramatic and expensive social impact. Previous studies have indicated that the blockade of two monoaminergic receptors, α1b-adrenergic and 5-HT2A, could inhibit the development of behavioral sensitization to drugs of abuse, a hallmark of drug-seeking and drug-taking behaviors in rodents. Here, in order to develop a potential therapeutic treatment of alcohol dependence in humans, we have blocked these two monoaminergic receptors by a combination of antagonists already approved by Health Agencies. We show that the association of ifenprodil (1 mg/kg) and cyproheptadine (1 mg/kg) (α1-adrenergic and 5-HT2 receptor antagonists marketed as Vadilex ® and Periactine ® in France, respectively) blocks behavioral sensitization to amphetamine in C57Bl6 mice and to alcohol in DBA2 mice. Moreover, this combination of antagonists inhibits alcohol intake in mice habituated to alcohol (10% v/v) and reverses their alcohol preference. Finally, in order to verify that the effect of ifenprodil was not due to its anti-NMDA receptors property, we have shown that a combination of prazosin (0.5 mg/kg, an α1b-adrenergic antagonist, Mini-Press ® in France) and cyproheptadine (1 mg/kg) could also reverse alcohol preference. Altogether these findings strongly suggest that combined prazosin and cyproheptadine could be efficient as a therapy to treat alcoholism in humans. Finally, because α1b-adrenergic and 5-HT2A receptors blockade also inhibits behavioral sensitization to psychostimulants, opioids and tobacco, it cannot be excluded that this combination will exhibit some efficacy in the treatment of addiction to other abused drugs.

D-amphetamine improves attention performance in adolescent Wistar, but not in SHR rats, in a two-choice visual discrimination task.

The validity of spontaneous hypertensive rat (SHR) as a model of attention deficit hyperactivity disorder (ADHD) has been explored by comparing SHR with Wistar rats in a test of attention, the two-choice visual discrimination task (2-CVDT). Animals were 4-5 weeks old during the training phase of the experiment and 6-7 weeks old during the testing phase in which they were tested with D-amphetamine, a stimulant drug used for the treatment of ADHD. As compared to Wistar, SHR showed a slightly better attention performance, a slightly lower impulsivity level, and a lower general activity during the training phase, but these differences disappeared or lessened thereafter, during the testing phase. D-amphetamine (0.5, 1 mg/kg) improved attention performance in Wistar, but not in SHR, and did not modify impulsivity and activity in the two strains. In conclusion, the present study did not demonstrate that SHR represents a valid model of ADHD, since it did not show face validity regarding the behavioral symptoms of ADHD and predictive validity regarding the effect of a compound used for the treatment of ADHD. On the other hand, this study showed that the 2-CVDT may represent a suitable tool for evaluating in adolescent Wistar rats the effect on attention of compounds intended for the treatment of ADHD.

Caspase-1 activation by NLRP3 inflammasome dampens IL-33-dependent house dust mite-induced allergic lung inflammation.

The cysteine protease caspase-1 (Casp-1) contributes to innate immunity through the assembly of NLRP3, NLRC4, AIM2, and NLRP6 inflammasomes. Here we ask whether caspase-1 activation plays a regulatory role in house dust mite (HDM)-induced experimental allergic airway inflammation. We report enhanced airway inflammation in caspase-1-deficient mice exposed to HDM with a marked eosinophil recruitment, increased expression of IL-4, IL-5, IL-13, as well as full-length and bioactive IL-33. Furthermore, mice deficient for NLRP3 failed to control eosinophil influx in the airways and displayed augmented Th2 cytokine and chemokine levels, suggesting that the NLPR3 inflammasome complex controls HDM-induced inflammation. IL-33 neutralization by administration of soluble ST2 receptor inhibited the enhanced allergic inflammation, while administration of recombinant IL-33 during challenge phase enhanced allergic inflammation in caspase-1-deficient mice. Therefore, we show that caspase-1, NLRP3, and ASC, but not NLRC4, contribute to the upregulation of allergic lung inflammation. Moreover, we cannot exclude an effect of caspase-11, because caspase-1-deficient mice are deficient for both caspases. Mechanistically, absence of caspase-1 is associated with increased expression of IL-33, uric acid, and spleen tyrosine kinase (Syk) production. This study highlights a critical role of caspase-1 activation and NLPR3/ASC inflammasome complex in the down-modulation of IL-33 in vivo and in vitro, thereby regulating Th2 response in HDM-induced allergic lung inflammation.

Myotonic dystrophy CTG expansion affects synaptic vesicle proteins, neurotransmission and mouse behaviour.

Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology.

Absence of TI-VAMP/Vamp7 leads to increased anxiety in mice.

Vesicular (v)- and target (t)-SNARE proteins assemble in SNARE complex to mediate membrane fusion. Tetanus neurotoxin-insensitive vesicular-associated membrane protein (TI-VAMP/VAMP7), a vesicular SNARE expressed in several cell types including neurons, was previously shown to play a major role in exocytosis involved in neurite growth in cultured neurons. Here we generated a complete constitutive knock-out by deleting the exon 3 of Vamp7. Loss of TI-VAMP expression did not lead to any striking developmental or neurological defect. Knock-out mice displayed decreased brain weight and increased third ventricle volume. Axon growth appeared normal in cultured knock-out neurons. Behavioral characterization unraveled that TI-VAMP knock-out was associated with increased anxiety. Our results thus suggest compensatory mechanisms allowing the TI-VAMP knock-out mice to fulfill major developmental processes. The phenotypic traits unraveled here further indicate an unexpected role of TI-VAMP-mediated vesicular traffic in anxiety and suggest a role for TI-VAMP in higher brain functions.

Effects of atomoxetine, desipramine, d-amphetamine and methylphenidate on impulsivity in juvenile rats, measured in a T-maze procedure.

Impulsivity is a core symptom of Attention Deficit/Hyperactivity Disorder (ADHD). In the present study, we assessed the effects of two stimulants, methylphenidate and d-amphetamine and of two non stimulant noradrenaline reuptake inhibitors, atomoxetine and desipramine, on the tolerance to delay of reward, taken as an index of impulsivity, in juvenile Wistar rats. Animals were trained in a T-maze to choose between a small-and-immediate reward and a large-but-30s-delayed reward. The effects of drugs were studied on the performance of animals at 30-40 day of age. Methylphenidate (3mg/kg), atomoxetine (1mg/kg), d-amphetamine (1 and 2mg/kg) and desipramine (8 and 16mg/kg) increased the number of choices of the large-but-delayed reward, i.e. decreased impulsivity. Given that these drugs are commonly prescribed in ADHD, these data indicate that the T-maze procedure in juvenile animals may be suitable for testing the therapeutic potential of drugs intended to the treatment of ADHD in children.

A new mouse model for the trisomy of the Abcg1-U2af1 region reveals the complexity of the combinatorial genetic code of down syndrome.

Mental retardation in Down syndrome (DS), the most frequent trisomy in humans, varies from moderate to severe. Several studies both in human and based on mouse models identified some regions of human chromosome 21 (Hsa21) as linked to cognitive deficits. However, other intervals such as the telomeric region of Hsa21 may contribute to the DS phenotype but their role has not yet been investigated in detail. Here we show that the trisomy of the 12 genes, found in the 0.59 Mb (Abcg1-U2af1) Hsa21 sub-telomeric region, in mice (Ts1Yah) produced defects in novel object recognition, open-field and Y-maze tests, similar to other DS models, but induces an improvement of the hippocampal-dependent spatial memory in the Morris water maze along with enhanced and longer lasting long-term potentiation in vivo in the hippocampus. Overall, we demonstrate the contribution of the Abcg1-U2af1 genetic region to cognitive defect in working and short-term recognition memory in DS models. Increase in copy number of the Abcg1-U2af1 interval leads to an unexpected gain of cognitive function in spatial learning. Expression analysis pinpoints several genes, such as Ndufv3, Wdr4, Pknox1 and Cbs, as candidates whose overexpression in the hippocampus might facilitate learning and memory in Ts1Yah mice. Our work unravels the complexity of combinatorial genetic code modulating different aspect of mental retardation in DS patients. It establishes definitely the contribution of the Abcg1-U2af1 orthologous region to the DS etiology and suggests new modulatory pathways for learning and memory.

Progressive loss of dopaminergic neurons in the ventral midbrain of adult mice heterozygote for Engrailed1: a new genetic model for Parkinson's disease?

Engrailed1 is a developmental gene of the homeogene family that controls the survival of midbrain dopaminergic neurons throughout life. Since these neurons have been crucially implicated in Parkinson's disease (PD), transgenic mice lacking one En1 allele could be of particular interest for the development of an animal model for PD. We showed in En1+/- mice, some traits reminiscent of PD such as (1) a progressive loss of mesencephalic dopaminergic (DA) neurons, and (2) motor deficits, anhedonia, decreased social interactions and depression-like behaviours. Further validation is needed, but these first results suggest that En1+/- mice could provide a promising model for the study of PD.

Methylphenidate reduces impulsive behaviour in juvenile Wistar rats, but not in adult Wistar, SHR and WKY rats.

RATIONALE: Impulsivity is a core symptom of attention deficit/hyperactivity disorder (ADHD). The spontaneously hypertensive rats (SHR) is a strain commonly used as an animal model of ADHD. However, there is no clear evidence that psychostimulants, which are used for treatment of ADHD, reduce impulsivity in SHR. Because ADHD mainly affects children, it may be relevant to study psychostimulants on juvenile animals. OBJECTIVES: Using tolerance to delay of reward as index of impulsivity, the effects of methylphenidate were assessed in adult SHR, Wistar Kyoto (WKY) and Wistar rats and in juvenile Wistar rats. MATERIALS AND METHODS: Animals were trained in a T-maze to choose between a small-but-immediate and a large-but-delayed reward. Adult SHR, WKY and Wistar rats were compared for their ability to tolerate a 15-s delay. The effect of methylphenidate on the tolerance to a 30-s delay was studied in adult rats of the three strains and in juvenile (4.5 to 6.5-week-old) Wistar rats. RESULTS: In adult rats, the waiting ability was lower in SHR than in control strains. Waiting ability was improved by methylphenidate (3 and 5 mg/kg) in juveniles, but not by methylphenidate (3 mg/kg) in adults. CONCLUSIONS: These data support the idea that SHR are more impulsive than control strains. However, at the dose studied, methylphenidate fails to improve tolerance to delay in adult rats whatever the strain used. The reduction of impulsivity induced by methylphenidate in juvenile Wistar rats indicates that juvenile animals may be suitable for testing the therapeutic potential of drugs intended to the treatment of ADHD in children.

Progressive loss of dopaminergic neurons in the ventral midbrain of adult mice heterozygote for Engrailed1.

Engrailed1 and Engrailed2 (En1 and En2) are two developmental genes of the homeogene family expressed in the developing midbrain. En1 and, to a lesser degree, En2 also are expressed in the adult substantia nigra (SN) and ventral tegmental area (VTA), two dopaminergic (DA) nuclei of the ventral midbrain. In an effort to study En1/2 adult functions, we have analyzed the phenotype of mice lacking one En1 allele in an En2 wild-type context. We show that in this mutant the number of DA neurons decreases slowly between 8 and 24 weeks after birth to reach a stable 38 and 23% reduction in the SN and VTA, respectively, and that neuronal loss can be antagonized by En2 recombinant protein infusions in the midbrain. These loss and gain of function experiments firmly establish that En1/2 is a true survival factor for DA neurons in vivo. Neuronal death in the mutant is paralleled by a 37% decrease in striatal DA, with no change in serotonin content. Using established protocols, we show that, compared with their wild-type littermates, En1+/- mice have impaired motor skills, an anhedonic-like behavior, and an enhanced resignation phenotype; they perform poorly in social interactions. However, these mice do not differ from their wild-type littermates in anxiety-measuring tests. Together, these results demonstrate that En1/2 genes have important adult physiological functions. They also suggest that mice lacking only one En1 allele could provide a novel model for the study of diseases associated with progressive DA cell death.

Chronic treatment with sulbutiamine improves memory in an object recognition task and reduces some amnesic effects of dizocilpine in a spatial delayed-non-match-to-sample task.

The effect of a sulbutiamine chronic treatment on memory was studied in rats with a spatial delayed-non-match-to-sample (DNMTS) task in a radial maze and a two trial object recognition task. After completion of training in the DNMTS task, animals were subjected for 9 weeks to daily injections of either saline or sulbutiamine (12.5 or 25 mg/kg). Sulbutiamine did not modify memory in the DNMTS task but improved it in the object recognition task. Dizocilpine, impaired both acquisition and retention of the DNMTS task in the saline-treated group, but not in the two sulbutiamine-treated groups, suggesting that sulbutiamine may counteract the amnesia induced by a blockade of the N-methyl-D-aspartate glutamate receptors. Taken together, these results are in favor of a beneficial effect of sulbutiamine on working and episodic memory.

Strain differences in sucrose preference and in the consequences of unpredictable chronic mild stress.

Effects of unpredictable chronic mild stress (UCMS) on anhedonic-like behaviour, physical state, body weight, learning and memory were investigated in three strains of mice. These strains were chosen among 11 strains that were tested in a first experiment for their sucrose consumption and preference for sucrose solutions of different concentrations. In the second experiment, groups of mice of the CBA/H, C57BL/6 and DBA/2 strains were submitted to 7 weeks of UCMS. Measures of the sucrose consumption, the evaluation of the physical state and the measurement of body weight were weekly assessed. Following 4-week period of UCMS, sub-groups of stressed and non-stressed mice were submitted to the spontaneous alternation test in the Y-maze, and then to the water-maze test for spatial learning and memory. UCMS induced a significant decrease of the sucrose consumption in CBA/H and in C57BL/6 but not in DBA/2 mice. The UCMS effect on sucrose intake in CBA/H mice was associated with a body weight loss and a physical state degradation. Spatial learning in a water maze was not disturbed by UCMS, however, a long-term memory impairment was observed in CBA/H stressed mice during a probe test. In the Y-maze, UCMS did not modify spontaneous alternation. These results show both an anhedonic-like and an amnesic effect of UCMS in CBA/H mice. They also reveal a difference of sensitivity to UCMS according to the strain of mice.

Prenatal exposure of rats to Ginkgo biloba extract (EGb 761) increases neuronal survival/growth and alters gene expression in the developing fetal hippocampus.

Hippocampal neuron survival/growth and gene expression have been examined after prenatal (in utero) exposure of rats to EGb 761, a leaf extract of Ginkgo biloba. Oral administration of EGb 761 (100 or 300 mg/kg/day) to pregnant dams for 5 days increased the number of hippocampal neurons (maintained in culture) of their fetuses, indicating a neurotrophic effect of the extract. Using large-scale oligonucleotide microarrays containing over 8000 combined rat genes and expressed sequence tag clusters, it was shown that treatment of pregnant dams with EGb 761 (25, 50 or 100 mg/kg/day for 5 days) altered the expression of 187 genes in the hippocampi of male fetuses and 160 genes in those of female fetuses. Using gene-cluster analysis, these genes were grouped into 18 distinct clusters for males and 17 distinct clusters for females. Among these clusters, 35 genes shared a common expression pattern in male and female hippocampal development. Of these genes, the changes observed in insulin growth factor II, insulin growth factor binding protein 2, testosterone repressed prostate message-2, glutathione-dependent dehydroascorbate reductase, lipoprotein lipase, guanylate cyclase and DNA binding protein Brn-2 were confirmed by real-time quantitative polymerase chain reaction. These findings, which have provided the first genetic profile of the effects of EGb 761 on the developing rat hippocampus, increase our understanding of the molecular and genetic programs that are activated by the extract. These effects of EGb 761 may underlie its neuroprotective properties.

Alpha1b-adrenergic receptors control locomotor and rewarding effects of psychostimulants and opiates.

Drugs of abuse, such as psychostimulants and opiates, are generally considered as exerting their locomotor and rewarding effects through an increased dopaminergic transmission in the nucleus accumbens. Noradrenergic transmission may also be implicated because most psychostimulants increase norepinephrine (NE) release, and numerous studies have indicated interactions between noradrenergic and dopaminergic neurons through alpha1-adrenergic receptors. However, analysis of the effects of psychostimulants after either destruction of noradrenergic neurons or pharmacological blockade of alpha1-adrenergic receptors led to conflicting results. Here we show that the locomotor hyperactivities induced by d-amphetamine (1-3 mg/kg), cocaine (5-20 mg/kg), or morphine (5-10 mg/kg) in mice lacking the alpha1b subtype of adrenergic receptors were dramatically decreased when compared with wild-type littermates. Moreover, behavioral sensitizations induced by d-amphetamine (1-2 mg/kg), cocaine (5-15 mg/kg), or morphine (7.5 mg/kg) were also decreased in knock-out mice when compared with wild-type. Ruling out a neurological deficit in knock-out mice, both strains reacted similarly to novelty, to intraperitoneal saline, or to the administration of scopolamine (1 mg/kg), an anti-muscarinic agent. Finally, rewarding properties could not be observed in knock-out mice in an oral preference test (cocaine and morphine) and conditioned place preference (morphine) paradigm. Because catecholamine tissue levels, autoradiography of D1 and D2 dopaminergic receptors, and of dopamine reuptake sites and locomotor response to a D1 agonist showed that basal dopaminergic transmission was similar in knock-out and wild-type mice, our data indicate a critical role of alpha1b-adrenergic receptors and noradrenergic transmission in the vulnerability to addiction.