Striatal-enriched protein tyrosine phosphatase controls responses to aversive stimuli: implication for ethanol drinking.

The STriatal-Enriched protein tyrosine Phosphatase (STEP) is a brain-specific phosphatase whose dysregulation in expression and/or activity is associated with several neuropsychiatric disorders. We recently showed that long-term excessive consumption of ethanol induces a sustained inhibition of STEP activity in the dorsomedial striatum (DMS) of mice. We further showed that down-regulation of STEP expression in the DMS, and not in the adjacent dorsolateral striatum, increases ethanol intake, suggesting that the inactivation of STEP in the DMS contributes to the development of ethanol drinking behaviors. Here, we compared the consequence of global deletion of the STEP gene on voluntary ethanol intake to the consumption of an appetitive rewarding substance (saccharin) or an aversive solution (quinine or denatonium). Whereas saccharin intake was similar in STEP knockout (KO) and wild type (WT) littermate mice, the consumption of ethanol as well as quinine and denatonium was increased in STEP KO mice. These results suggested that the aversive taste of these substances was masked upon deletion of the STEP gene. We therefore hypothesized that STEP contributes to the physiological avoidance towards aversive stimuli. To further test this hypothesis, we measured the responses of STEP KO and WT mice to lithium-induced conditioned place aversion (CPA) and found that whereas WT mice developed lithium place aversion, STEP KO mice did not. In contrast, conditioned place preference (CPP) to ethanol was similar in both genotypes. Together, our results indicate that STEP contributes, at least in part, to the protection against the ingestion of aversive agents

Striatal-enriched protein tyrosine phosphatase controls responses to aversive stimuli: implication for ethanol drinking.

The STriatal-Enriched protein tyrosine Phosphatase (STEP) is a brain-specific phosphatase whose dysregulation in expression and/or activity is associated with several neuropsychiatric disorders. We recently showed that long-term excessive consumption of ethanol induces a sustained inhibition of STEP activity in the dorsomedial striatum (DMS) of mice. We further showed that down-regulation of STEP expression in the DMS, and not in the adjacent dorsolateral striatum, increases ethanol intake, suggesting that the inactivation of STEP in the DMS contributes to the development of ethanol drinking behaviors. Here, we compared the consequence of global deletion of the STEP gene on voluntary ethanol intake to the consumption of an appetitive rewarding substance (saccharin) or an aversive solution (quinine or denatonium). Whereas saccharin intake was similar in STEP knockout (KO) and wild type (WT) littermate mice, the consumption of ethanol as well as quinine and denatonium was increased in STEP KO mice. These results suggested that the aversive taste of these substances was masked upon deletion of the STEP gene. We therefore hypothesized that STEP contributes to the physiological avoidance towards aversive stimuli. To further test this hypothesis, we measured the responses of STEP KO and WT mice to lithium-induced conditioned place aversion (CPA) and found that whereas WT mice developed lithium place aversion, STEP KO mice did not. In contrast, conditioned place preference (CPP) to ethanol was similar in both genotypes. Together, our results indicate that STEP contributes, at least in part, to the protection against the ingestion of aversive agents

Saccharin consumption is similar in STEP KO and WT mice.

<p>STEP WT and KO mice were submitted to a continuous access two-bottle choice paradigm with access to one bottle of a saccharin solution and one bottle of tap water. Saccharin concentration was increased every 4 days (0.005, 0.015, 0.033 and 0.066%). Results are expressed as mean ± SEM of (<b>A</b>) saccharin or (<b>B</b>) total fluid intake per 24 hours for each saccharin concentration. Two-way RM-ANOVA showed no effect of genotype for <b>A</b> [<i>F</i>(1,15) = .357, <i>p</i> = .559] and <b>B</b> [<i>F</i>(1,15) = .508, <i>p</i> = .487], an effect of saccharin concentration for <b>A</b> [<i>F</i>(3,45) = 72.3, <i>p</i> <.001] and <b>B</b> [<i>F</i>(3,45) = 17.207, <i>p</i> <.001] and no interaction between genotype and saccharin concentration for <b>A</b> [<i>F</i>(3,45) = .0441, <i>p</i> = .988] and <b>B</b> [<i>F</i>(3,45) = .284, <i>p</i> = .837]. n = 8-9.</p

Quinine and denatonium consumption is increased in STEP KO vs. WT mice.

<p>STEP WT and KO mice were submitted to a continuous access two-bottle choice paradigm with access to one bottle of a quinine or denatonium solution and one bottle of tap water. Quinine or denatonium concentration was increased every 4 days (0.01, 0.03, 0.06, 0.12 and 0.24 mM for quinine and 0.03, 0.06, 0.12 and 0.24 mM for denatonium). Results are expressed as mean ± SEM of (<b>A</b>) quinine or (<b>C</b>) denatonium intake or total fluid intake per 24 hours for each (<b>B</b>) quinine or (<b>D</b>) denatonium concentration. Two-way RM-ANOVA showed an effect of genotype for <b>A</b> [<i>F</i>(1,15) = 7.524, <i>p</i> = .015] and <b>C</b> [<i>F</i>(1,16) = 4.594, <i>p</i> = .048] but not for <b>B</b> [<i>F</i>(1,15) = 1.181, <i>p</i> = .294] and <b>D</b> [<i>F</i>(1,16) = 0.365, <i>p</i> = .554], an effect of concentration for <b>A</b> [<i>F</i>(4,60) = 64.307 <i>p</i> <.001], <b>B</b> [<i>F</i>(4,60) = 24.150, <i>p</i> <.001], <b>C</b> [<i>F</i>(3,48) = 19.434, <i>p</i> <.001] and <b>D</b> [<i>F</i>(3,48) = 10.080, <i>p</i> <.001], and no interaction between genotype and concentration for <b>A</b> [<i>F</i>(4,60) = 2.435, <i>p</i> = .057], <b>B</b> [<i>F</i>(4,60) = 1.290, <i>p</i> = .284], <b>C</b> [<i>F</i>(3,48) = 2.462, <i>p</i> = .074] and <b>D</b> [<i>F</i>(3,48) = 1.007, <i>p</i> = .398]. *<i>p</i> <.05 vs. WT, method of contrasts. n = 8–9.</p

STEP KO mice do not express conditioned place aversion to lithium chloride.

<p>STEP WT and KO mice were submitted to a conditioned place aversion paradigm. The conditioning phase consisted in 3 days of daily administration (s.c.) of saline (mornings) or 130 mg/kg lithium chloride (LiCl; afternoons) solution followed by the confinement in the saline- or LiCl-paired compartment for 45 min (3 saline and 3 LiCl conditioning sessions). One day after the third conditioning day, a 20-min postconditioning test was conducted. Results are expressed as mean ± SEM of the percentage of time spent in the LiCl-paired compartment during the preconditioning and postconditioning tests. Two-way ANOVA conducted on the postconditioning values showed an effect of genotype [<i>F</i>(1,28) = 4.784, <i>p</i> = .037], no effect of conditioning [<i>F</i>(1,28) = 2.395, <i>p =</i> .133] and no interaction between genotype and conditioning [<i>F</i>(1,28) = 2.460, <i>p</i> = .128]. *<i>p</i> <.05 vs. Saline, ^#<i>p</i> <.05 vs. KO, method of contrasts. n = 7–9.</p

Global deletion of STEP does not alter spontaneous locomotor activity.

<p>Results are expressed as mean ± SEM of distance traveled (cm) per 5-min bins in STEP WT and KO mice during a 30-min session. Two-way RM-ANOVA showed an effect of time [<i>F</i>(5,60) = 49.091, <i>p</i> <.001] but no effect genotype [<i>F</i>(1,12) = 1.161, <i>p</i> = .302] and no interaction between time and genotype [<i>F</i>(5,60) = .355, <i>p</i> = .877]. n = 6–8.</p

Conditioned place preference to ethanol is not altered in STEP KO mice.

<p>STEP WT and KO mice were submitted to an ethanol-induced conditioned place preference paradigm. The conditioning phase consisted in 8 days of daily administration (i.p.) of either saline (Sal) or 2.0 g/kg ethanol (EtOH) solution followed by the confinement in the saline- or ethanol-paired compartment for 5 min (4 saline and 4 ethanol conditioning sessions). One day after the eighth session, a 30-min postconditioning test was conducted. Results are expressed as mean ± SEM of the percentage of time spent in the ethanol-paired compartment during the preconditioning and postconditioning tests. Two-way ANOVA conducted on the postconditioning values showed no effect of genotype [<i>F</i>(1,33) = .168, <i>p</i> = .684], an effect of conditioning [<i>F</i>(1,33) = 18.587, <i>p <</i>.001.] and no interaction between genotype and conditioning [<i>F</i>(1,33) = .330, <i>p</i> = .570.]. *<i>p</i> <.05, **<i>p</i> <.01 vs. Saline, method of contrasts. n = 8–10.</p

Suitability of PER.C6 (R) cells to generate epidemic and pandemic influenza vaccine strains by reverse genetics

Reverse genetics, the generation of influenza viruses from cDNA. presents a rapid method for creating vaccine strains. The technique necessitates the use of cultured cells. Due to technical and regulatory requirements, the choice of cell lines for production of human influenza vaccines is limited. PER.C6 (R) cells, among the most extensively characterized and documented cells, support growth of all influenza Viruses tested to date, and can be grown to high densities in large bioreactors in the absence of serum or micro carriers. Here, the suitability of these cells for the generation of influenza Viruses by reverse genetics was investigated. A range of viruses reflective of vaccine strains was rescued exclusively using PER.C6 cells by Various transfection methods, including an animal component-free procedure. Furthermore, a whole inactivated vaccine carrying the HA and NA segments of A/HK/156/97 (H5N1) that was both rescued from and propagated oil PER.C6 cells, conferred protection in a mouse model. Thus PER.C6 cells provide an attractive platform for generation of influenza vaccine strains via reverse genetics