Increased ethanol intake and preference in cyclin D2 knockout mice

Inhibitory effects of passive ethanol exposure on brain neurogenesis have been extensively documented in animal models. In contrast, a role of brain neurogenesis in ethanol self-administration has not been addressed, as yet. The aim of this study was to assess intake of, and preference for, ethanol solutions [2-16% (v/v)] in a mouse model of adult neurogenesis deficiency based on permanent knockout (KO) of cyclin D2 (Ccnd2). Wild type (WT) and Ccnd2 KO mice did not differ in 2% and 4% ethanol intake. The KO group consumed significantly more ethanol in g/kg when offered with 8% or 16% ethanol as compared with the WT controls. The WT and KO mice did not differ in 2% ethanol preference, but the KO group showed a significantly higher preference for 4-16% ethanol. Animal and human studies have suggested that the low level of response to the sedative/hypnotic effects of alcohol is genetically associated with enhanced alcohol consumption. However, in this study, there were no between-genotype differences in ethanol-induced loss of righting reflex. Previous reports have also suggested that high ethanol intake is genetically associated with the avidity for sweets and better acceptance of bitter solutions. However, the KO and WT mice consumed similar amounts of saccharin solutions and the KOs consumed less quinine (i.e. bitter) solutions as compared with the WTs. In conclusion, these results may indicate that Ccnd2 and, possibly, brain neurogenesis are involved in central regulation of ethanol intake in mice.     

Taok2 controls behavioral response to ethanol in mice

Despite recent advances in the understanding of ethanol's biological action, many of the molecular targets of ethanol and mechanisms behind ethanol's effect on behavior remain poorly understood. In an effort to identify novel genes, the products of which regulate behavioral responses to ethanol, we recently identified a mutation in the dtao gene that confers resistance to the locomotor stimulating effect of ethanol in Drosophila. dtao encodes a member of the Ste20 family of serine/threonine kinases implicated in MAP kinase signaling pathways. In this study, we report that conditional ablation of the mouse dtao homolog, Taok2, constitutively and specifically in the nervous system, results in strain‐specific and overlapping alterations in ethanol‐dependent behaviors. These data suggest a functional conservation of dtao and Taok2 in mediating ethanol's biological action and identify Taok2 as a putative candidate gene for ethanol use disorders in humans.     

Progress in a replicated selection for elevated blood ethanol concentrations in HDID mice

Drinking in the dark (DID) is a limited access ethanol‐drinking phenotype in mice. High Drinking in the Dark (HDID‐1) mice have been bred for 27 selected generations (S27) for elevated blood ethanol concentrations (BECs) after a 4‐h period of access to 20% ethanol. A second replicate line (HDID‐2) was started later from the same founder population and is currently in S20. An initial report of response to selection in HDID‐1 was published after S11. This article reports genetic and behavioral characteristics of both lines in comparison with the HS controls. Heritability is low in both replicates (h² = 0.09) but the lines have shown 4–5 fold increases in BEC since S0; 80% of HDID‐1 and 60% of HDID‐2 mice reach BECs greater than 1.0 mg/ml. Several hours after a DID test, HDID mice show mild signs of withdrawal. Although not considered during selection, intake of ethanol (g/kg) during the DID test increased by approximately 80% in HDID‐1 and 60% in HDID‐2. Common genetic influences were more important than environmental influences in determining the similarity between BEC and intake for HDID mice. Analysis of the partitioning of intake showed that 60% of intake is concentrated in the last 2 h of the 4 h session. However, this has not changed during selection. Hourly BECs during the DID test reach peak levels after 3 or 4 h of drinking. HDID mice do not differ from HS mice in their rate of elimination of an acute dose of alcohol .     

Loss of RAB-3/A in Caenorhabditis elegans and the mouse affects behavioral response to ethanol

The mechanisms by which ethanol induces changes in behavior are not well understood. Here, we show that Caenorhabditis elegans loss-of-function mutations in the synaptic vesicle-associated RAB-3 protein and its guanosine triphosphate exchange factor AEX-3 confer resistance to the acute locomotor effects of ethanol. Similarly, mice lacking one or both copies of Rab3A are resistant to the ataxic and sedative effects of ethanol, and Rab3A haploinsufficiency increases voluntary ethanol consumption. These data suggest a conserved role of RAB-3-/RAB3A-regulated neurotransmitter release in ethanol-related behaviors.     

Increased ethanol resistance and consumption in Eps8 knockout mice correlates with altered actin dynamics

Dynamic modulation of the actin cytoskeleton is critical for synaptic plasticity, abnormalities of which are thought to contribute to mental illness and addiction. Here we report that mice lacking Eps8, a regulator of actin dynamics, are resistant to some acute intoxicating effects of ethanol and show increased ethanol consumption. In the brain, the N-methyl-D-aspartate (NMDA) receptor is a major target of ethanol. We show that Eps8 is localized to postsynaptic structures and is part of the NMDA receptor complex. Moreover, in Eps8 null mice, NMDA receptor currents and their sensitivity to inhibition by ethanol are abnormal. In addition, Eps8 null neurons are resistant to the actin-remodeling activities of NMDA and ethanol. We propose that proper regulation of the actin cytoskeleton is a key determinant of cellular and behavioral responses to ethanol.     

Saccharomyces cerevisiae strains with different degrees of ethanol tolerance exhibit different adaptive responses to produced ethanol

Two Saccharomyces cerevisiae strains with different degrees of ethanol tolerance adapted differently to produced ethanol. Adaptation in the less ethanol-tolerant strain was high and resulted in a reduced formation of ethanol-induced respiratory deficient mutants and an increased ergosterol content of the cells. Adaptation in the more ethanol-tolerant strain was less pronounced. Journal of Industrial Microbiology & Biotechnology (2000) 24, 75–78. Received 22 June 1999/ Accepted in revised form 06 October 1999     

Deletion of agouti-related protein blunts ethanol self-administration and binge-like drinking in mice

The melanocortin (MC) system is composed of peptides that are cleaved from the polypeptide precursor proopiomelanocortin (POMC). Recent pharmacological and genetic evidence suggests that melanocortin receptor (MCR) signaling modulates neurobiological responses to ethanol and ethanol intake. Agouti-related protein (AgRP) is synthesized by neurons in the arcuate nucleus of the hypothalamus and is a natural antagonist of MCRs. Because central administration of the functionally active AgRP fragment AgRP-(83–132) increases ethanol intake by C57BL/6 J mice, we determined if mutant mice lacking normal production of AgRP (AgRP^−/−) and maintained on a C57BL/6 J genetic background would show reduced self-administration of ethanol relative to littermate wild-type (AgRP^+/+) mice. AgRP^−/— mice showed reduced 8% (v/v) ethanol-reinforced lever-pressing behavior relative to AgRP^+/+ mice in daily 2-h sessions, but normal sucrose-, saccharin- and water-reinforced lever-pressing. Similarly, AgRP^−/− mice showed reduced consumption of 8% ethanol in a two-bottle limited access test (2 h/day), although this effect was largely sex-dependent. Using drinking-in-the-dark (DID) procedures, AgRP^−/— mice showed blunted binge-like drinking of 20% (v/v) ethanol which was associated with lower blood ethanol levels (85 mg/dl) relative to AgRP^+/+ mice (133 mg/dl) after 4 h of intake. AgRP^−/− mice showed normal ethanol metabolism and did not show altered sensitivity to the sedative effects of ethanol. These observations with genetically altered mice are consistent with previous pharmacological data and suggest that endogenous AgRP signaling modulates the reinforcing properties of ethanol and binge-like ethanol drinking.     

Mouse inbred strain differences in ethanol drinking to intoxication

Recently, we described a simple procedure, Drinking in the Dark (DID), in which C57BL/6J mice self-administer ethanol to a blood ethanol concentration (BEC) above 1 mg/ml. The test consists of replacing the water with 20% ethanol in the home cage for 4 h early during the dark phase of the light/dark cycle. Three experiments were conducted to explore this high ethanol drinking model further. In experiment 1, a microanalysis of C57BL/6J behavior showed that the pattern of ethanol drinking was different from routine water intake. In experiment 2, drinking impaired performance of C57BL/6J on the accelerating rotarod and balance beam. In experiment 3, 12 inbred strains were screened to estimate genetic influences on DID and correlations with other traits. Large, reliable differences in intake and BEC were detected among the strains, with C57BL/6J showing the highest values. Strain means were positively correlated with intake and BEC in the standard (24 h) and a limited (4 h) two-bottle ethanol vs. water test, but BECs reached higher levels for DID. Strain mean correlations with other traits in the Mouse Phenome Project database supported previously reported genetic relationships of high ethanol drinking with low chronic ethanol withdrawal severity and low ethanol-conditioned taste aversion. We extend these findings by showing that the correlation estimates remain relatively unchanged even after correcting for phylogenetic relatedness among the strains, thus relaxing the assumption that the strain means are statistically independent. We discuss applications of the model for finding genes that predispose pharmacologically significant drinking in mice.     

Taste responses to sweet stimuli in alpha-gustducin knockout and wild-type mice

The importance of alpha-gustducin in sweet taste transduction is based on data obtained with sucrose and the artificial sweetener SC45647. Here we studied the role of alpha-gustducin in sweet taste. We compared the behavioral and electrophysiological responses of alpha-gustducin knockout (KO) and wild-type (WT) mice to 11 different sweeteners, representing carbohydrates, artificial sweeteners, and sweet amino acids. In behavioral experiments, over 48-h preference ratios were measured in two-bottle preference tests. In electrophysiological experiments, integrated responses of chorda tympani (CT) and glossopharyngeal (NG) nerves were recorded. We found that preference ratios of the KO mice were significantly lower than those of WT for acesulfame-K, dulcin, fructose, NC00174, D-phenylalanine, L-proline, D-tryptophan, saccharin, SC45647, sucrose, but not neotame. The nerve responses to all sweeteners, except neotame, were smaller in the KO mice than in the WT mice. The differences between the responses in WT and KO mice were more pronounced in the CT than in the NG. These data indicate that alpha-gustducin participates in the transduction of the sweet taste in general.     

Hepatic microvascular responses to ischemia-reperfusion in low-density lipoprotein receptor knockout mice

The overall objective of this study was to determine whether genetically induced hypercholesterolemia alters the inflammatory and microvascular responses of mouse liver to ischemia-reperfusion (I/R). The accumulation of rhodamine 6G-labeled leukocytes and the number of nonperfused sinusoids (NPS) were monitored (by intravital microscopy) in the liver of wild-type (WT) and low-density lipoprotein receptor-deficient (LDLr(-/-)) mice for 1 h after a 30-min period of normothermic ischemia. Plasma alanine transaminase (ALT) levels were used to monitor hepatocellular injury. Microvascular leukostasis as well as increases in NPS and plasma ALT were observed at 60 min after hepatic I/R in both WT and in LDLr(-/-) mice; however, these responses were greatly exaggerated in LDLr(-/-) mice. Pretreatment of LDLr(-/-) mice with gadolinium chloride, which reduces Kupffer cell function, attenuated the hepatic leukostasis, NPS, and hepatocellular injury elicited by I/R. Similar protection against I/R was observed in LDLr(-/-) mice pretreated with antibodies directed against tumor necrosis factor-alpha, intercellular adhesion molecule-1 (ICAM-1), or P-selectin. These findings indicate that chronic hypercholesterolemia predisposes the hepatic microvasculature to the deleterious effects of I/R. Kupffer cell activation and the leukocyte adhesion receptors ICAM-1 and P-selectin appear to contribute to the exaggerated inflammatory responses observed in the postischemic liver of LDLr(-/-) mice.     

Calcium responses to thyrotropin-releasing hormone, gonadotropin-releasing hormone and somatostatin in phospholipase css3 knockout mice

These studies examined the importance of phospholipase Cbeta (PLCbeta) in the calcium responses of pituitary cells using PLCbeta3 knockout mice. Pituitary tissue from wild-type mice contained PLCbeta1 and PLCbeta3 but not PLCbeta2 or PLCbeta4. Both Galphaq/11 and Gbetagamma can activate PLCbeta3, whereas only Galphaq/11 activates PLCss1 effectively. In knockout mice, PLCbeta3 was absent, PLCbeta1 was not up-regulated, and PLCbeta2 and PLCbeta4 were not expressed. Since somatostatin inhibited influx of extracellular calcium in pituitary cells from wild-type and PLCbeta3 knockout mice, the somatostatin signal pathway was intact. However, somatostatin failed to increase intracellular calcium in pituitary cells from either wild-type or knockout mice under a variety of conditions, indicating that it did not stimulate PLCbeta3. In contrast, somatostatin increased intracellular calcium in aortic smooth muscle cells from wild-type mice, although it evoked no calcium response in cells from PLCbeta3 knockout animals These results show that somatostatin, like other Gi/Go-linked hormones, can stimulate a calcium transient by activating PLCbeta3 through Gbetagamma, but this response does not normally occur in pituitary cells. The densities of Gi and Go, as well as the relative concentrations of PLCbeta1 and PLCbeta3, were similar in cells that responded to somatostatin with an increase in calcium and pituitary cells. Calcium responses to 1 nM and 1 microM TRH and GnRH were identical in pituitary cells from wild-type and PLCbeta3 knockout mice, as were responses to other Gq-linked agonists. These results show that in pituitary cells, PLCbeta1 is sufficient to transmit signals from Gq-coupled hormones, whereas PLCbeta3 is required for the calcium-mobilizing actions of somatostatin observed in smooth muscle cells.     

Attraction of red turpentine beetle and other Scolytinae to ethanol, 3‐carene or ethanol + 3‐carene in an Oregon pine forest

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Reduced hypothermic and hypnotic responses to ethanol in PACAP-deficient mice

Using pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient mice, we investigated whether PACAP is involved in the intoxicating effects of ethanol. The structure of PACAP is highly conserved during evolution, and in Drosophila, loss-of-function mutations in a PACAP-like neuropeptide gene, amnesiac, result in impairment of memory retention and increased sensitivity to ethanol. In mice, PACAP deficiency is associated with impaired memory performance and hippocampal long-term potentiation (LTP), however, sensitivity to ethanol has not been well investigated. Here, we addressed this issue in our recently developed PACAP-deficient mice. Sleep time (duration of the loss of righting reflex) was markedly shortened in PACAP-deficient mice compared with wild-type, although latency to the loss of righting reflex was not different between the two groups. Ethanol-induced hypothermia in wild-type control mice was significantly reduced in PACAP-deficient mice. Blood ethanol levels were not different between the two groups, excluding the possibility of increased ethanol metabolism. Thus, in contrast to that in Drosophila, PACAP deficiency in mammals caused a reduced sensitivity to ethanol. However, in both cases, PACAP or amnesiac products are likely to play significant roles in modifying the intoxicating effects of ethanol.     

An evolutionary conserved role for anaplastic lymphoma kinase in behavioral responses to ethanol

Anaplastic lymphoma kinase (Alk) is a gene expressed in the nervous system that encodes a receptor tyrosine kinase commonly known for its oncogenic function in various human cancers. We have determined that Alk is associated with altered behavioral responses to ethanol in the fruit fly Drosophila melanogaster, in mice, and in humans. Mutant flies containing transposon insertions in dAlk demonstrate increased resistance to the sedating effect of ethanol. Database analyses revealed that Alk expression levels in the brains of recombinant inbred mice are negatively correlated with ethanol-induced ataxia and ethanol consumption. We therefore tested Alk gene knockout mice and found that they sedate longer in response to high doses of ethanol and consume more ethanol than wild-type mice. Finally, sequencing of human ALK led to the discovery of four polymorphisms associated with a low level of response to ethanol, an intermediate phenotype that is predictive of future alcohol use disorders (AUDs). These results suggest that Alk plays an evolutionary conserved role in ethanol-related behaviors. Moreover, ALK may be a novel candidate gene conferring risk for AUDs as well as a potential target for pharmacological intervention.     

Increased apoptosis during neonatal brain development underlies the adult behavioral deficits seen in mice lacking a functional paternally expressed gene 3 (Peg3)

Inactivation of the maternally imprinted, paternally expressed gene 3 (Peg3) induces deficits in olfactory function, sexual and maternal behaviors, oxytocin neuron number, metabolic homeostasis and growth. Peg3 is expressed in a number of developing hypothalamic and basal forebrain structures and is a component of the P53 apoptosis pathway. Peg3 inactivation in neuronal cell culture lines inhibits P53 mediated apoptosis, which is important in the early postnatal development and sexual differentiation of the brain. In this study, we investigated the effect of inactivating the Peg3 gene on the incidence of caspase 3 positive cells (a marker of apoptosis) in 4‐ and 6‐day postpartum mouse brain. Inactivating the Peg3 gene resulted in an increase in the incidence of total forebrain caspase 3 positive cells at 4 and 6 days postpartum. Increases in specific neuroanatomical regions including the bed nucleus of the stria terminalis, nucleus accumbens, caudate putamen, medial pre‐optic area, arcuate nucleus, medial amygdala, anterior cortical and posteriodorsal amygdaloid nuclei, were also observed. In wild‐type mice, sex differences in the incidence of caspase 3 positive cells in the medial amygdala, bed nucleus of the stria terminalis, nucleus accumbens, arcuate nucleus and the M2 motor cortex, were also observed. This neural sex difference was ameliorated in the Peg‐3 mutant. These findings suggest that the neuronal and behavioral deficits seen in mice lacking a functional Peg3 gene are mediated by increases in the incidence of early neonatal apoptosis in neuroanatomical regions important for reproductive behavior, olfactory and pheromonal processing, thermoregulation and reward. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Ceramide metabolism analysis in a model of binge drinking reveals both neuroprotective and toxic effects of ethanol

Binge drinking is a common form of alcohol abuse that involves repeated rounds of intoxication followed by withdrawal. The episodic effects of binge drinking and withdrawal on brain resident cells are thought to contribute to neural remodeling and neurological damage. However, the molecular mechanisms for these neurodegenerative effects are not understood. Ethanol (EtOH) regulates the metabolism of ceramide, a highly bioactive lipid that is enriched in brain. We used a mouse model of binge drinking to determine the effects of EtOH intoxication and withdrawal on brain ceramide metabolism. Intoxication and acute alcohol withdrawal were each associated with distinct changes in ceramide regulatory genes and metabolic products. EtOH intoxication was accompanied by decreased concentrations of multiple ceramides, coincident with reductions in the expression of enzymes involved in the production of ceramides, and increased expression of ceramide‐degrading enzymes. EtOH withdrawal was associated with specific increases in ceramide C16:0, C18:0, and C20:0 and increased expression of enzymes involved with ceramide production. These data suggest that EtOH intoxication may evoke a ceramide phenotype that is neuroprotective, whereas EtOH withdrawal results in a metabolic shift that increases the production of potentially toxic ceramide species.

     

Increased entropy production in diaphragm muscle of PPAR alpha knockout mice

Peroxisome proliferator activated receptor alpha (PPAR alpha) regulates fatty acid beta-oxidation (FAO) and plays a central role in the metabolic and energetic homeostasis of striated muscles. The thermodynamic consequences of the absence of PPAR alpha were investigated in diaphragm muscle of PPAR alpha knockout mice (KO). Statistical mechanics provides a powerful tool for determining entropy production, which quantifies irreversible chemical processes generated by myosin molecular motors and which is the product of thermodynamic force A/T (chemical affinity A and temperature T) and thermodynamic flow (myosin crossbridge (CB) cycle velocity upsilon). The behavior of both wild type (WT) and KO diaphragm was shown to be near-equilibrium and in a stationary state, but KO was farther from equilibrium than WT. In KO diaphragm, a substantial decrease in contractile function was associated with an increase in both A/T and upsilon and with profound histological injuries such as contraction band necrosis. There were no changes in PPAR delta and gamma expression levels or myosin heavy chain (MHC) patterns. In KO diaphragm, a marked increase in entropy production (A/T x upsilon) accounted for major thermodynamic dysfunction and a dramatic increase in irreversible chemical processes during the myosin CB cycle.     

Attenuated renovascular constrictor responses to angiotensin II in adenosine 1 receptor knockout mice

In the present experiments we examined the renovascular constrictor effects of ANG II in the chronic and complete absence of A1 adenosine receptors (A1AR) using mice with targeted deletion of the A1AR gene. Glomerular filtration rate (GFR) was not different between A1AR +/+ and A1AR -/- mice under control conditions (450.5 +/- 60 vs. 475.2 +/- 62.5 microl/min) but fell significantly less in A1AR -/- mice during infusion of ANG II at 1.5 ng/min (A1AR +/+: 242 +/- 32.5 microl/min, A1AR -/-: 371 +/- 42 microl/min; P = 0.03). Bolus injection of 1, 10, and 100 ng of ANG II reduced renal blood flow and increased renal vascular resistance significantly more in A1AR +/+ than in A1AR -/- mice. Perfused afferent arterioles isolated from A1AR +/+ mice constricted in response to bath ANG II with an EC50 of 1.5 +/- 0.4 x 10(-10) mol/l, whereas a right shift in the dose-response relationship with an EC50 of 7.3 +/- 1.2 x 10(-10) mol/l (P < 0.05) was obtained in arterioles from A1AR -/- mice (P < 0.05). The expression of AT1A receptor mRNA was not different in kidney RNA from A1AR +/+ or A1AR -/- mice. We conclude that chronic A1AR deficiency diminishes the effectiveness of ANG II to constrict renal resistance vessels and to reduce GFR.     

Divergent coronary flow responses to uridine adenosine tetraphosphate in atherosclerotic ApoE knockout mice

Uridine adenosine tetraphosphate (Up₄A) exerts potent relaxation in porcine coronary arteries that is reduced following myocardial infarction, suggesting a crucial role for Up₄A in the regulation of coronary flow (CF) in cardiovascular disorders. We evaluated the vasoactive effects of Up₄A on CF in atherosclerosis using ApoE knockout (KO) mice ex vivo and in vivo. Functional studies were conducted in isolated mouse hearts using the Langendorff technique. Immunofluorescence was performed to assess purinergic P2X₁ receptor (P2X₁R) expression in isolated mouse coronary arteries. In vivo effects of Up₄A on coronary blood flow (CBF) were assessed using ultrasound. Infusion of Up₄A (10^?9–10^?5 M) into isolated mouse hearts resulted in a concentration-dependent reduction in CF in WT and ApoE KO mice to a similar extent; this effect was exacerbated in ApoE KO mice fed a high-fat diet (HFD). The P2X₁R antagonist MRS2159 restored Up₄A-mediated decreases in CF more so in ApoE KO + HFD than ApoE KO mice. The smooth muscle to endothelial cell ratio of coronary P2X₁R expression was greater in ApoE KO + HFD than ApoE KO or WT mice, suggesting a net vasoconstrictor potential of P2X₁R in ApoE KO + HFD mice. In contrast, Up₄A (1.6 mg/kg) increased CBF to a similar extent among the three groups. In conclusion, Up₄A decreases CF more in ApoE KO + HFD mice, likely through a net upregulation of vasoconstrictor P2X₁R. In contrast, Up₄A increases CBF in vivo regardless of the atherosclerotic model.