Different attentional dysfunctions in eEF2K−/−, IL1RAPL1−/− and SHANK3Δ11−/− mice

A common feature of several psychiatric disorders is the attentional impairment. eEF2K ^?/?, IL1RAPL1 ^?/? and SHANK3Δ11 ^?/? mice were used as animal models consistently linked to changes in synaptic plasticity, learning and memory. All knockout (KO) mice and their corresponding littermates were submitted to the novel object recognition (NOR) and visual object recognition (VOR) tasks. In the NOR, eEF2K^?/? mice exhibited a normal performance in terms of mean discrimination index, while SHANK3Δ11^?/? and IL1RAPL1 ^?/? mice were impaired when a delay of 2 and 24 hours was introduced. Surprisingly, when submitted to VOR, where the two objects were replaced with two shapes delivered from two iPods, all the mutant mice performed worse than those in the NOR. In VOR, the application of motion to different shapes, to increase attention, improved performance in eEF2K ^?/? and IL1RAPL1 ^?/? but not in SHANK3Δ11 ^?/? mice. In SHANK3Δ11 ^?/? mice, attentional deficit was also present even if different motions were applied to the same shapes or when these mice were repeatedly exposed for 5 days to the context. Behavioral analysis showed that eEF2K^?/? and IL1RAPL1 ^?/? mice had a good flexibility tested in the T‐maze. eEF2K^?/? showed normal self‐grooming. On the basis of previous literature data indicating that SHANK3Δ11 ^?/? showed impaired flexibility and reduced sociability, we identified in this genotype the most exhaustive model showing all the core symptoms of autism spectrum disorder including a heavy visual attention deficit. These findings show the importance of VOR to identify mouse models of autism.     

Involvement of A2A receptors in anxiolytic,locomotor and motivational properties of ethanol in mice

We have shown previously that mice lacking the adenosine A_2A receptor (A_2AR) generated on a CD1 background self‐administer more ethanol and exhibit hyposensitivity to acute ethanol. We aimed to investigate if the increased propensity of A_2A^?/? mice to consume ethanol is associated with an altered sensitivity in the motivational properties of ethanol in the conditioned place preference (CPP) and conditioned taste aversion (CTA) paradigms and with an altered development of sensitization to the locomotor effects of ethanol. We also tested their sensitivity to the anxiolytic effects of ethanol. Our results show that A_2A^?/? mice produced on a CD1 background displayed a reduced ethanol‐induced CPP and an increased sensitivity to the anxiolytic and locomotor‐stimulant effects of ethanol, but they did not show alteration in ethanol‐induced CTA and locomotor sensitization. Ethanol‐induced CPP, ethanol consumption and the locomotor effects of ethanol were also tested in A_2A^?/? mice produced on a C57BL/6J background. Our results emphasized the importance of the genetic background because alteration in ethanol consumption and preference, ethanol‐induced CPP and locomotor‐stimulant effects were not found in knockout mice produced on the alcohol‐preferring C57BL/6J genetic background. Finally, the A_2AR agonist, 2‐p‐(2‐carboxyethyl)‐phenylethylamino‐5′‐N‐ethylcarboxamidoadenosine hydrochloride (CGS 21680), reduced ethanol consumption and preference in C57BL/6J mice. In conclusion, A_2AR deficiency in mice generated on a CD1 background leads to high ethanol consumption that is associated with an increased sensitivity to the locomotor‐stimulant/anxiolytic effects of ethanol and a decrease in ethanol‐induced CPP.     

Chronic voluntary ethanol intake hypersensitizes 5‐HT1A autoreceptors in C57BL/6J mice

Alcoholism is a complex disorder involving, among others, the serotoninergic (5‐HT) system, mainly regulated by 5‐HT_1A autoreceptors in the dorsal raphe nucleus. 5‐HT_1A autoreceptor desensitization induced by chronic 5‐HT reuptake inactivation has been associated with a decrease in ethanol intake in mice. We investigated here whether, conversely, chronic ethanol intake could induce 5‐HT_1A autoreceptor supersensitivity, thereby contributing to the maintenance of high ethanol consumption. C57BL/6J mice were subjected to a progressive ethanol intake procedure in a free‐choice paradigm (3–10% ethanol versus tap water; 21 days) and 5‐HT_1A autoreceptor functional state was assessed using different approaches. Acute administration of the 5‐HT_1A receptor agonist ipsapirone decreased the rate of tryptophan hydroxylation in striatum, and this effect was significantly larger (+75%) in mice that drank ethanol than in those drinking water. Furthermore, ethanol intake produced both an increased potency (+45%) of ipsapirone to inhibit the firing of 5‐HT neurons, and a raise (+35%) in 5‐HT_1A autoreceptor‐mediated stimulation of [³⁵S]GTP‐γ‐S binding in the dorsal raphe nucleus. These data showed that chronic voluntary ethanol intake in C57BL/6J mice induced 5‐HT_1A autoreceptor supersensitivity, at the origin of a 5‐HT neurotransmission deficit, which might be causally related to the addictive effects of ethanol intake.     

Enhanced dopamine function in DISC1‐L100P mutant mice: implications for schizophrenia

Significant advances have been made in understanding the role of disrupted‐in‐schizophrenia‐1 (DISC1) in the brain and accumulating findings suggest the possible implication of DISC1 in the regulation of dopamine (DA) function. A mutation in the second exon of DISC1 at L100P leads to the development of schizophrenia‐related behavior in mutant mice (DISC1‐L100P). We investigated here the role of DA in the expression of schizophrenia‐related endophenotypes in the DISC1‐L100P genetic mouse model. The mutated DISC1 resulted in facilitation of the psychostimulant effect of amphetamine in DISC1‐L100P mutant mice assessed in the open field and prepulse inhibition (PPI) tests. Biochemical studies detected a 2.1‐fold increase in the proportion of striatal D receptors without significant changes in DA release in vivo in the striatum of DISC1‐L100P mutants in response to the low dose of amphetamine. The D₂ receptor antagonist haloperidol reversed the hyperactivity, PPI and latent inhibition (LI) deficits and blocked the psychostimulant effect of amphetamine in DISC1‐L100P mutants. Taken together, our findings show the role of DISC1 in D₂‐related pathophysiological mechanism of schizophrenia, linking DISC1 with well‐established DA hypothesis of schizophrenia.     

Distinct serum proteome profiles associated with collagen‐induced arthritis and complete Freund's adjuvant‐induced inflammation in CD38−/− mice: The discriminative power of protein species or proteoforms

Collagen‐type‐II‐induced arthritis (CIA) is an autoimmune disease, which involves a complex host systemic response including inflammatory and autoimmune reactions. CIA is milder in CD38^?/? than in wild‐type (WT) mice. ProteoMiner‐equalized serum samples were subjected to 2D‐DiGE and MS‐MALDI‐TOF/TOF analyses to identify proteins that changed in their relative abundances in CD38^?/? versus WT mice either with arthritis (CIA⁺), with no arthritis (CIA^?), or with inflammation (complete Freund's adjuvant (CFA)‐treated mice). Multivariate analyses revealed that a multiprotein signature (n = 28) was able to discriminate CIA⁺ from CIA^? mice, and WT from CD38^?/? mice within each condition. Likewise, a distinct multiprotein signature (n = 16) was identified which differentiated CIA⁺ CD38^?/? mice from CIA⁺ WT mice, and lastly, a third multiprotein signature (n = 18) indicated that CD38^?/? and WT mice could be segregated in response to CFA treatment. Further analyses showed that the discriminative power to distinguish these groups was reached at protein species level and not at the protein level. Hence, the need to identify and quantify proteins at protein species level to better correlate proteome changes with disease processes. It is crucial for plasma proteomics at the low‐abundance protein species level to apply the ProteoMiner enrichment. All MS data have been deposited in the ProteomeXchange with identifiers PXD001788, PXD001799 and PXD002071 ( http://proteomecentral.proteomexchange.org/dataset/PXD001788 , http://proteomecentral.proteomexchange.org/dataset/PXD001799 and http://proteomecentral.proteomexchange.org/dataset/PXD002071 ).     

Increased fear learning,spatial learning as well as neophobia in Rgs2−/− mice

Anxiety disorders result from a complex interplay of genetic and environmental factors such as stress. On the level of cellular signaling, regulator of G protein signaling 2 (Rgs2) has been implicated in human and rodent anxiety. However, there is limited knowledge about the role of Rgs2 in fear learning and reactivity to stress. In this study, Rgs2^?/? mice showed increased fear learning, male mice displayed increased contextual and cued fear learning, while females showed selectively enhanced cued fear learning. Male Rgs2^?/? mice displayed increased long‐term‐contextual fear memory, but increased cued fear extinction. Learning in spatial non‐aversive paradigms was also increased in Rgs2^?/? mice. Female, but not male mice show increased spatial learning in the Barnes maze, while male mice showed enhanced place preference in the IntelliCage, rendering enhanced cognitive function non‐specific for aversive stimuli. Consistent with the previous results, Rgs2 deletion resulted in increased innate anxiety, including neophobic behavior expressed as hypolocomotion, in three different tests based on the approach‐avoidance conflict. Acute electric foot shock stress provoked hypolocomotion in several exploration‐based tests, suggesting fear generalization in both genotypes. Rgs2 deletion was associated with reduced monoaminergic neurotransmitter levels in the hippocampus and prefrontal cortex and disturbed corresponding GPCR expression of the adrenergic, serotonergic, dopaminergic and neuropeptide Y system. Taken together, Rgs2 deletion promotes improved cognitive function as well as increased anxiety‐like behavior, but has no effect on acute stress reactivity. These effects may be related to the observed disruption of the monoaminergic systems.     

AGC1‐malate aspartate shuttle activity is critical for dopamine handling in the nigrostriatal pathway

The mitochondrial transporter of aspartate‐glutamate Aralar/AGC1 is a regulatory component of the malate‐aspartate shuttle. Aralar deficiency in mouse and human causes a shutdown of brain shuttle activity and global cerebral hypomyelination. A lack of neurofilament‐labeled processes is detected in the cerebral cortex, but whether different types of neurons are differentially affected by Aralar deficiency is still unknown. We have now found that Aralar‐knockout (Aralar‐KO) post‐natal mice show hyperactivity, anxiety‐like behavior, and hyperreactivity with a decrease of dopamine (DA) in terminal‐rich regions. The striatum is the brain region most affected in terms of size, amino acid and monoamine content. We find a decline in vesicular monoamine transporter‐2 (VMAT2) levels associated with increased DA metabolism through MAO activity (DOPAC/DA ratio) in Aralar‐KO striatum. However, no decrease in DA or in the number of nigral tyrosine hydroxylase‐positive cells was detected in Aralar‐KO brainstem. Adult Aralar‐hemizygous mice presented also increased DOPAC/DA ratio in striatum and enhanced sensitivity to amphetamine. Our results suggest that Aralar deficiency causes a fall in GSH/GSSG ratio and VMAT2 in striatum that might be related to a failure to produce mitochondrial NADH and to an increase of reactive oxygen species (ROS) in the cytosol. The results indicate that the nigrostriatal dopaminergic system is a target of Aralar deficiency.     

Bryostatin‐1 ameliorated experimental colitis in Il‐10−/− Mice by protecting the intestinal barrier and limiting immune dysfunction

Bryostatin‐1 (Bry‐1) has been proven to be effective and safe in clinical trials of a variety of immune‐related diseases. However, little is known about its effect on Crohn's disease (CD). We aimed to investigate the impact of Bry‐1 on CD‐like colitis and determine the mechanism underlying this effect. In the present study, 15‐week‐old male Il‐10^?/? mice with spontaneous colitis were divided into positive control and Bry‐1‐treated (Bry‐1, 30 μg/kg every other day, injected intraperitoneally for 4 weeks) groups. Age‐matched, male wild‐type (WT) mice were used as a negative control. The effects of Bry‐1 on colitis, intestinal barrier function and T cell responses as well as the potential regulatory mechanisms were evaluated. We found that the systemic delivery of Bry‐1 significantly ameliorated colitis in Il‐10^?/? mice, as demonstrated by decreases in the disease activity index (DAI), inflammatory score and proinflammatory mediator levels. The protective effects of Bry‐1 on CD‐like colitis included the maintenance of intestinal barrier integrity and the helper T cell (Th)/regulatory T cell (Treg) balance. These effects of Bry‐1 may act in part through nuclear factor erythroid 2‐related factor 2 (Nrf2) signalling activation and STAT3/4 signalling inhibition. The protective effect of Bry‐1 on CD‐like colitis suggests Bry‐1 has therapeutic potential in human CD, particularly given the established clinical safety of Bry‐1.     

Lysosomal cholesterol accumulation in macrophages leading to coronary atherosclerosis in CD38−/− mice

The disruption in transportation of oxLDL‐derived cholesterol and the subsequent lipid accumulation in macrophages are the hallmark events in atherogenesis. Our recent studies demonstrated that lysosomal Ca²⁺ messenger of nicotinic acid adenine dinucleotide phosphate (NAADP), an enzymatic product of CD38 ADP‐ribosylcyclase (CD38), promoted lipid endocytic trafficking in human fibroblast cells. The current studies are designed to examine the functional role of CD38/NAADP pathway in the regulation of lysosomal cholesterol efflux in atherosclerosis. Oil red O staining showed that oxLDL concentration‐dependently increased lipid buildup in bone marrow‐derived macrophages from both wild type and CD38^?/?, but to a significant higher extent with CD38 gene deletion. Bodipy 493/503 fluorescence staining found that the deposited lipid in macrophages was mainly enclosed in lysosomal organelles and largely enhanced with the blockade of CD38/NAADP pathway. Filipin staining and direct measurement of lysosome fraction further revealed that the free cholesterol constituted a major portion of the total cholesterol segregated in lysosomes. Moreover, in situ assay disclosed that both lysosomal lumen acidity and the acid lipase activity were reduced upon cholesterol buildup in lysosomes. In CD38^?/? mice, treatment with Western diet (12 weeks) produced atherosclerotic damage in coronary artery with striking lysosomal cholesterol sequestration in macrophages. These data provide the first experimental evidence that the proper function of CD38/NAADP pathway plays an essential role in promoting free cholesterol efflux from lysosomes and that a defection of this signalling leads to lysosomal cholesterol accumulation in macrophages and results in coronary atherosclerosis in CD38^?/? mice.     

Immunohistochemical biomarkers and distribution of telocytes in ApoE−/− mice

Telocytes had been identified as a peculiar stromal cell type implicated in tissue homeostasis and the development and pathophysiology of diseases. Telocyte existed in most organs and tissues in humans and animals. However, few studies have examined telocytes in ApoE gene deficient mice. In our studies, we verified the existence, the morphology and immunohistochemical characteristics of telocytes in critical organs of the ApoE^?/? mice. Male adult ApoE^?/? mice were selected as an experimental model. Immunohistochemical bio‐markers, such as CD34, CD117, CD28, Vimentin and PDGFR‐α were utilized to determine the distribution and morphology of telocytes in the heart, liver and kidney. Telocyte expressed positively for CD34 and CD117, and partial telocyte and telopode expressed positively for PDGFR‐α in heart and liver, but negatively in kidney. Double immunofluorescence assays for CD28/Vimentin, CD34/CD117 and CD34/PDGFR‐α were used to demonstrate the biochemistry speciality of telocytes, respectively. The evidence of telocytes in the ApoE^‐/‐ mice is the first step of our sturdy, which aims to demonstrate changes in telocytes in atherosclerosis in this animal model.     

Deletion of the Kv2.1 delayed rectifier potassium channel leads to neuronal and behavioral hyperexcitability

The Kv2.1 delayed rectifier potassium channel exhibits high‐level expression in both principal and inhibitory neurons throughout the central nervous system, including prominent expression in hippocampal neurons. Studies of in vitro preparations suggest that Kv2.1 is a key yet conditional regulator of intrinsic neuronal excitability, mediated by changes in Kv2.1 expression, localization and function via activity‐dependent regulation of Kv2.1 phosphorylation. Here we identify neurological and behavioral deficits in mutant (Kv2.1^?/?) mice lacking this channel. Kv2.1^?/? mice have grossly normal characteristics. No impairment in vision or motor coordination was apparent, although Kv2.1^?/? mice exhibit reduced body weight. The anatomic structure and expression of related Kv channels in the brains of Kv2.1^?/? mice appear unchanged. Delayed rectifier potassium current is diminished in hippocampal neurons cultured from Kv2.1^?/? animals. Field recordings from hippocampal slices of Kv2.1^?/? mice reveal hyperexcitability in response to the convulsant bicuculline, and epileptiform activity in response to stimulation. In Kv2.1^?/? mice, long‐term potentiation at the Schaffer collateral – CA1 synapse is decreased. Kv2.1^?/? mice are strikingly hyperactive, and exhibit defects in spatial learning, failing to improve performance in a Morris Water Maze task. Kv2.1^?/? mice are hypersensitive to the effects of the convulsants flurothyl and pilocarpine, consistent with a role for Kv2.1 as a conditional suppressor of neuronal activity. Although not prone to spontaneous seizures, Kv2.1^?/? mice exhibit accelerated seizure progression. Together, these findings suggest homeostatic suppression of elevated neuronal activity by Kv2.1 plays a central role in regulating neuronal network function .     

AnxA5 reduces plaque inflammation of advanced atherosclerotic lesions in apoE−/− mice

Annexin A5 (AnxA5) exerts anti‐inflammatory, anticoagulant and anti‐apoptotic effects through binding cell surface expressed phosphatidylserine. The actions of AnxA5 on atherosclerosis are incompletely understood. We investigated effects of exogenous AnxA5 on plaque morphology and phenotype of advanced atherosclerotic lesions in apoE^?/? mice. Advanced atherosclerotic lesions were induced in 12 weeks old Western type diet fed apoE^?/? mice using a collar placement around the carotid artery. After 5 weeks mice were injected either with AnxA5 (n = 8) or vehicle for another 4 weeks. AnxA5 reduced plaque macrophage content both in the intima (59% reduction, P < 0.05) and media (73% reduction, P < 0.01) of advanced atherosclerotic lesions of the carotid artery. These findings corroborated with advanced lesions of the aortic arch, where a 67% reduction in plaque macrophage content was observed with AnxA5 compared to controls (P < 0.01). AnxA5 did not change lesion extension, plaque apoptosis, collagen content, smooth muscle cell content or acellular plaque composition after 4 weeks of treatment as determined by immunohistochemistry in advanced carotid lesions. In vitro, AnxA5 exhibited anti‐inflammatory effects in macrophages and a flow chamber based assay demonstrated that AnxA5 significantly inhibited capture, rolling, adhesion as well as transmigration of peripheral blood mononuclear cells on a TNF‐α‐activated endothelial cell layer. In conclusion, short‐term treatment with AnxA5 reduces plaque inflammation of advanced lesions in apoE^?/? mice likely through interfering with recruitment and activation of monocytes to the inflamed lesion site. Suppressing chronic inflammation by targeting exposed phosphatidylserine may become a viable strategy to treat patients suffering from advanced atherosclerosis.     

Essential role of NMDA receptor channel ε4 subunit (GluN2D) in the effects of phencyclidine, but not methamphetamine

Phencyclidine (PCP), a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, increases locomotor activity in rodents and causes schizophrenia-like symptoms in humans. Although activation of the dopamine (DA) pathway is hypothesized to mediate these effects of PCP, the precise mechanisms by which PCP induces its effects remain to be elucidated. The present study investigated the effect of PCP on extracellular levels of DA (DA(ex)) in the striatum and prefrontal cortex (PFC) using in vivo microdialysis in mice lacking the NMDA receptor channel ε1 or ε4 subunit (GluRε1 [GluN2A] or GluRε4 [GluN2D]) and locomotor activity. PCP significantly increased DA(ex) in wildtype and GluRε1 knockout mice, but not in GluRε4 knockout mice, in the striatum and PFC. Acute and repeated administration of PCP did not increase locomotor activity in GluRε4 knockout mice. The present results suggest that PCP enhances dopaminergic transmission and increases locomotor activity by acting at GluRε4.     

Mitochondrial PKC‐ε deficiency promotes I/R‐mediated myocardial injury via GSK3β‐dependent mitochondrial permeability transition pore opening

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)‐induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase‐2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross‐clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2^?/?) and wild‐type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin‐related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N‐acetylcysteine (NAC) or PKC‐δ shRNA treatment on glycogen synthase kinase‐3β (GSK‐3β) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2^?/? mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC‐ε translocation was lower in ALDH2^?/? mice than in WT mice, and PKC‐δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre‐treatment under I/R injury. In addition, PKC‐ε inhibition caused activation of caspase9, caspase3 and Drp1Ser⁶¹⁶ in response to I/R stress. Importantly, expression of phosphorylated GSK‐3β (inactive form) was lower in ALDH2^?/? mice than in WT mice, and both were increased by NAC pre‐treatment. I/R‐induced mitochondrial translocation of GSK‐3β was inhibited by PKC‐δ shRNA or NAC pre‐treatment. In addition, mitochondrial membrane potential (?Ψ_m) was reduced in ALDH2^?/? mice after I/R, which was partly reversed by the GSK‐3β inhibitor (SB216763) or PKC‐δ shRNA. Collectively, our data provide the evidence that abnormal PKC‐ε/PKC‐δ ratio promotes the activation of Drp1 signalling, caspase cascades and GSK‐3β‐dependent mPTP opening, which results in mitochondrial injury‐triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2^?/? mice following I/R stress.     

Altered synaptic plasticity and behavioral abnormalities in CNGA3‐deficient mice

The role of the cyclic nucleotide‐gated (CNG) channel CNGA3 is well established in cone photoreceptors and guanylyl cyclase‐D‐expressing olfactory neurons. To assess a potential function of CNGA3 in the mouse amygdala and hippocampus, we examined synaptic plasticity and performed a comparative analysis of spatial learning, fear conditioning and step‐down avoidance in wild‐type mice and CNGA3 null mutants (CNGA3^?/?). CNGA3^?/? mice showed normal basal synaptic transmission in the amygdala and the hippocampus. However, cornu Ammonis (CA1) hippocampal long‐term potentiation (LTP) induced by a strong tetanus was significantly enhanced in CNGA3^?/? mice as compared with their wild‐type littermates. Unlike in the hippocampus, LTP was not significantly altered in the amygdala of CNGA3^?/? mice. Enhanced hippocampal LTP did not coincide with changes in hippocampus‐dependent learning, as both wild‐type and mutant mice showed a similar performance in water maze tasks and contextual fear conditioning, except for a trend toward higher step‐down latencies in a passive avoidance task. In contrast, CNGA3^?/? mice showed markedly reduced freezing to the conditioned tone in the amygdala‐dependent cued fear conditioning task. In conclusion, our study adds a new entry on the list of physiological functions of the CNGA3 channel. Despite the dissociation between physiological and behavioral parameters, our data describe a so far unrecognized role of CNGA3 in modulation of hippocampal plasticity and amydgala‐dependent fear memory.     

Differential expression of VGLUT3 in laboratory mouse strains: Impact on drug‐induced hyperlocomotion and anxiety‐related behaviors

The atypical vesicular glutamate transporter VGLUT3 is present in subpopulations of GABAergic interneurons in the cortex and the hippocampus, in subgroups of serotoninergic neurons in raphe nuclei, and in cholinergic interneurons in the striatum. C56BL/6N mice that no longer express VGLUT3 (VGLUT3^?/?) display anxiety‐associated phenotype, increased spontaneous and cocaine‐induced locomotor activity and decreased haloperidol‐induced catalepsy. Inbred mouse strains differ markedly in their sensitivity to anxiety and behavioral responses elicited by drugs. The purpose of this study was to investigate strain differences in VGLUT3 expression levels and its potential correlates with anxiety and reward‐guided behaviors. Five inbred mouse lines were chosen according to their contrasted anxiety and drugs sensitivity: C57BL/6N, C3H/HeN, DBA/2J, 129/Sv, and BALB/c. VGLUT3 protein expression was measured in different brain areas involved in reward or mood regulation (such as the striatum, the hippocampus, and raphe nuclei) and genetic variations in Slc17a8, the gene encoding for VGLUT3, have been explored. These five inbred mouse strains express very different levels of VGLUT3, which cannot be attributed to the genetic variation of the Slc17a8 locus. Furthermore, mice behavior in the open field, elevated plus maze, spontaneous‐ and cocaine‐induced locomotor was highly heterogeneous and only partially correlated to VGLUT3 levels. These data highlight the fact that one single gene polymorphism could not account for VGLUT3 expression variations, and that region specific VGLUT3 expression level variations might play a key role in the modulation of discrete behaviors.     

The effect of Sirt1 deficiency on Ca2+ and Na+ regulation in mouse ventricular myocytes

This study addressed the hypothesis that cardiac Sirtuin 1 (Sirt1) deficiency alters cardiomyocyte Ca²⁺ and Na⁺ regulation, leading to cardiac dysfunction and arrhythmogenesis. We used mice with cardiac‐specific Sirt1 knockout (Sirt1^?/?). Sirt1^flox/flox mice were served as control. Sirt1^?/? mice showed impaired cardiac ejection fraction with increased ventricular spontaneous activity and burst firing compared with those in control mice. The arrhythmic events were suppressed by KN93 and ranolazine. Reduction in Ca²⁺ transient amplitudes and sarcoplasmic reticulum (SR) Ca²⁺ stores, and increased SR Ca²⁺ leak were shown in the Sirt1^?/? mice. Electrophysiological measurements were performed using patch‐clamp method. While L‐type Ca²⁺ current (I_{Ca, L}) was smaller in Sirt1^?/? myocytes, reverse‐mode Na⁺/Ca²⁺ exchanger (NCX) current was larger compared with those in control myocytes. Late Na⁺ current (I_{Na, L}) was enhanced in the Sirt1^?/? mice, alongside with elevated cytosolic Na⁺ level. Increased cytosolic and mitochondrial reactive oxygen species (ROS) were shown in Sirt1^?/? mice. Sirt1^?/? cardiomyocytes showed down‐regulation of L‐type Ca²⁺ channel α1c subunit (Cav1.2) and sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 2a (SERCA2a), but up‐regulation of Ca²⁺/calmodulin‐dependent protein kinase II and NCX. In conclusions, these findings suggest that deficiency of Sirt1 impairs the regulation of intracellular Ca²⁺ and Na⁺ in cardiomyocytes, thereby provoking cardiac dysfunction and arrhythmogenesis.     

Masking Responses to Light in Period Mutant Mice

Masking is an acute effect of an external signal on an overt rhythm and is distinct from the process of entrainment. In the current study, we investigated the phase dependence and molecular mechanisms regulating masking effects of light pulses on spontaneous locomotor activity in mice. The circadian genes, Period1 (Per1) and Per2, are necessary components of the timekeeping machinery and entrainment by light appears to involve the induction of the expression of Per1 and Per2 mRNAs in the suprachiasmatic nuclei (SCN). We assessed the roles of the Per genes in regulating masking by assessing the effects of light pulses on nocturnal locomotor activity in C57BL/6J Per mutant mice. We found that Per1^?/? and Per2^?/? mice had robust negative masking responses to light. In addition, the locomotor activity of Per1^?/?/Per2^?/? mice appeared to be rhythmic in the light-dark (LD) cycle, and the phase of activity onset was advanced (but varied among individual mice) relative to lights off. This rhythm persisted for 1 to 2 days in constant darkness in some Per1^?/?/Per2^?/? mice. Furthermore, Per1^?/?/Per2^?/? mice exhibited robust negative masking responses to light. Negative masking was phase dependent in wild-type mice such that maximal suppression was induced by light pulses at zeitgeber time 14 (ZT14) and gradually weaker suppression occurred during light pulses at ZT16 and ZT18. By measuring the phase shifts induced by the masking protocol (light pulses were administered to mice maintained in the LD cycle), we found that the phase responsiveness of Per mutant mice was altered compared to wild-types. Together, our data suggest that negative masking responses to light are robust in Per mutant mice and that the Per1^?/?/Per2^?/? SCN may be a light-driven, weak/damping oscillator. (Author correspondence: shin.yamazaki@vanderbilt.edu)     

Dopamine Transporter Loss in 6-OHDA Parkinson’s Model Is Unmet by Parallel Reduction in Dopamine Uptake

The dopamine transporter (DAT) regulates synaptic dopamine (DA) in striatum and modulation of DAT can affect locomotor activity. Thus, in Parkinson’s disease (PD), DAT loss could affect DA clearance and locomotor activity. The locomotor benefits of L-DOPA may be mediated by transport through monoamine transporters and conversion to DA. However, its impact upon DA reuptake is unknown and may modulate synaptic DA. Using the unilateral 6-OHDA rat PD model, we examined [³H]DA uptake dynamics in relation to striatal DAT and tyrosine hydroxylase (TH) protein loss compared with contralateral intact striatum. Despite >70% striatal DAT loss, DA uptake decreased only ∼25% and increased as DAT loss approached 99%. As other monoamine transporters can transport DA, we determined if norepinephrine (NE) and serotonin (5-HT) differentially modulated DA uptake in lesioned striatum. Unlabeled DA, NE, and 5-HT were used, at a concentration that differentially inhibited DA uptake in intact striatum, to compete against [³H]DA uptake. In 6-OHDA lesioned striatum, DA was less effective, whereas NE was more effective, at inhibiting [³H]DA uptake. Furthermore, norepinephrine transporter (NET) protein levels increased and desipramine was ∼two-fold more effective at inhibiting NE uptake. Serotonin inhibited [³H]DA uptake, but without significant difference between lesioned and contralateral striatum. L-DOPA inhibited [³H]DA uptake two-fold more in lesioned striatum and inhibited NE uptake ∼five-fold more than DA uptake in naïve striatum. Consequently, DA uptake may be mediated by NET when DAT loss is at PD levels. Increased inhibition of DA uptake by L-DOPA and its preferential inhibition of NE over DA uptake, indicates that NET-mediated DA uptake may be modulated by L-DOPA when DAT loss exceeds 70%. These results indicate a novel mechanism for DA uptake during PD progression and provide new insight into how L-DOPA affects DA uptake, revealing possible mechanisms of its therapeutic and side effect potential.