Duration Of Loss Of Righting Reflex Research Articles

Early-life inflammation increases ethanol consumption in adolescent male mice

Recent studies have demonstrated that stress during the critical windows of development can evoke a cascade of neurological changes that can result in neuropsychiatric disorders later in life. In this study, we examined the effect of early-life inflammation on ethanol consumption in adolescent mice. C57BL/6J mice were assigned to either the control or Lipopolysaccharide (LPS) group on postnatal day 14 (P14). In the latter group, LPS at a dose of 50 μg/kg was injected intraperitoneally. The mice were weaned at P21, and behavior tests were performed at P45. Ethanol consumption was assessed using a two-bottle choice drinking paradigm. Anxiety-like behaviors were assessed by marble burying test (MBT), open field (OF), and elevated plus maze (EPM). Ethanol-induced loss of righting reflex (LORR), hypothermia and ethanol metabolism were assessed to evaluate ethanol intoxication. P14 LPS-injected adolescent male mice exhibited significantly increased ethanol preference and consumption, with a similar taste preference for saccharin and avoidance of quinine. The adolescent male mice showed increased anxiety-like behaviors in the OF and EPM tests, and an increased duration of LORR, without affecting the hypothermic effects of ethanol and ethanol metabolism. Interestingly, these behavioral changes were not obvious in female mice. In conclusion, our data indicate that early-life inflammation may be a risk factor for ethanol consumption in adolescents with greater changes observed in male mice. Significance StatementOur study is the first preclinical model to report the enhancement effect of early-life inflammation on ethanol consumption in adolescent male mice and our findings provide a valuable mouse model to examine the neurobiological mechanisms mediating the long-lasting effects of early-life inflammation on alcohol use disorders vulnerability.

Preparation of Dihydromyricetin-Loaded Self-Emulsifying Drug Delivery System and Its Anti-Alcoholism Effect.

Intraperitoneal injection of dihydromyricetin (DMY) has shown promising potential in the treatment of alcoholism. However, its therapeutic effect is limited due to its low solubility, poor stability, and high gut-liver first-pass metabolism, resulting in very low oral bioavailability. In this study, we developed a DMY-loaded self-emulsifying drug delivery system (DMY-SEDDS) to enhance the oral bioavailability and anti-alcoholism effect of DMY. DMY-SEDDS improved the oral absorption of DMY by facilitating lymphatic transport. The area under the concentration-time curve (AUC) of DMY in the DMY-SEDDS group was 4.13-fold higher than in the DMY suspension group. Furthermore, treatment with DMY-SEDDS significantly enhanced the activities of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in the liver of mice (p < 0.05). Interestingly, DMY-SEDDS also increased ADH activity in the stomach of mice with alcoholism (p < 0.01), thereby enhancing ethanol metabolism in the gastrointestinal tract and reducing ethanol absorption into the bloodstream. As a result, the blood alcohol concentration of mice with alcoholism was significantly decreased after DMY-SEDDS treatment (p < 0.01). In the acute alcoholism mice model, compared to saline treatment, DMY-SEDDS prolonged the onset of LORR (loss of righting reflex) (p < 0.05) and significantly shortened the duration of LORR (p < 0.01). Additionally, DMY-SEDDS treatment significantly reduced gastric injury in acute alcoholism mice. Collectively, these findings demonstrate the potential of DMY-SEDDS as a treatment in the treatment of alcoholism.

G protein-coupled receptor 158 modulates sensitivity to the sedative-hypnotic effect of ethanol in male mice.

Sensitivity to ethanol provides an index of the predisposition to recover from unconsciousness induced by a dose of ethanol. The role of the G protein-coupled receptor 158 (GPR158) in modulating sensitivity to the sedative-hypnotic effect of ethanol has not been investigated. Loss of righting reflex (LORR) is a behavioral indicator of hypnosis in rodents. In this study, Gpr158-/- mice and wild-type (WT) littermates (n = 8/genotype) were tested using LORR induced by a dose of 3.5 g/kg ethanol, an open-field test (OFT), and a measure of blood ethanol concentration. The OFT was used to examine the role of GPR158 in the ethanol effect on motor activity in Gpr158-/- mice (n = 6/genotype). We also tested CamK2A-Cre;Gpr158fl/fl (n = 9) and Vgat-Cre;Gpr158fl/fl mice (n = 10) using the LORR test and OFT to compare with controls (n = 9 and 8, respectively). Gpr158 deficiency prolonged the LORR duration by 110.6%, t(14) = -5.241, p = 0.0001, without altering spontaneous activity, t(14) = -0.718, p = 0.485, or ethanol metabolism, F(1, 8) = 0.259, p = 0.625. Gpr158 knockout did not change the ethanol effect on locomotion, F(1, 10) = 0.262, p = 0.62. The LORR duration increased by 69% in the conditional knockouts of Gpr158 within calcium/calmodulin-dependent protein kinase II alpha-positive (CamK2A+ ) neurons, t(16) = -2.914, p = 0.01, and by 92% in the vesicular GABA transporter-positive (Vgat+ ) neurons, t(9.802) = -2.519, p = 0.023. Locomotion was not altered in Camk2A-Cre;Gpr158fl/fl , t(16) = 0.49, p = 0.631 or Vgat-Cre;Gpr158fl/fl mice, t(16) = 0.035, p = 0.972. This study reveals the key role of neuronal GPR158 in shaping sensitivity to the sedative-hypnotic effect of ethanol. These findings contribute to our understanding of the neurobiology of ethanol intoxication.

Microglia modulate general anesthesia through P2Y12 receptor

General anesthesia (GA) is an unconscious state produced by anesthetic drugs, which act on neurons to cause overall suppression of neuronal activity in the brain. Recent studies have revealed that GA also substantially enhances the dynamics of microglia, the primary brain immune cells, with increased process motility and territory surveillance. However, whether microglia are actively involved in GA modulation remains unknown. Here, we report a previously unrecognized role for microglia engaging in multiple GA processes. We found that microglial ablation reduced the sensitivity of mice to anesthetics and substantially shortened duration of loss of righting reflex (LORR) or unconsciousness induced by multiple anesthetics, thereby promoting earlier emergence from GA. Microglial repopulation restored the regular anesthetic recovery, and chemogenetic activation of microglia prolonged the duration of LORR. In addition, anesthesia-accompanying analgesia and hypothermia were also attenuated after microglial depletion. Single-cell RNA sequencing analyses showed that anesthesia prominently affected the transcriptional levels of chemotaxis and migration-related genes in microglia. By pharmacologically targeting different microglial motility pathways, we found that blocking P2Y12 receptor (P2Y12R) reduced the duration of LORR of mice. Moreover, genetic ablation of P2Y12R in microglia also promoted quicker recovery in mice from anesthesia, verifying the importance of microglial P2Y12R in anesthetic regulation. Our work presents the first evidence that microglia actively participate in multiple processes of GA through P2Y12R-mediated signaling and expands the non-immune roles of microglia in the brain.

Positive effect of ethanol-induced Lactococcus lactis on alcohol metabolism in mice

It is well known that exposure to environmental stresses could enhance the adaptability of bacteria and up-regulate the expression of a variety of oxidative stress-related genes and antioxidant enzymes. It is unclear whether the adaptability of microorganisms formed naturally in special environments could transfer to other organisms. The study aimed to evaluate the effects of untreated and ethanol-induced Lactococcus lactis intracellular extracts (U-IE and E-IE) on alcohol metabolism in mice. The positive effects of E-IE on alcohol metabolism in mice were revealed by the enhanced latency of loss of righting reflex (LORR), the reduced duration of LORR, the decrease of blood alcohol concentration, as well as the elevation of alcohol dehydrogenase (ADH) activities in the stomach and liver tissues. Furthermore, the potential benefits of E-IE on the liver were evaluated by biochemical parameters including the activities of serum transaminase, the levels of antioxidant enzymes, and the pathological changes of liver tissue. The present work put forward a new point that appropriate ethanol stress could enhance the intracellular ADH activity of L. lactis, and its intracellular extracts could continue to enhance alcohol metabolism in mice.

Pre-treatment with Tandospirone attenuates fentanyl-induced respiratory depression without affecting the analgesic effects of fentanyl in rodents

Opioid analgesics are widely used to treat acute, postoperative, and chronic pain. However, opioid receptor activation can result in severe respiratory depression. In this study, we demonstrated that Tandospirone (TS), a selective serotonin-1A receptor partial agonist, is effective against opioid-induced respiratory depression. Fentanyl was used to establish a respiratory depression model in rodents. We observed the effects of TS on respiratory depression in rats by using plethysmographic recordings and arterial oxygen saturation. In addition, we evaluated the effects of TS on fentanyl-induced sedation and analgesia by using the loss of righting reflex (LORR) and hot-plate tests, respectively. Rats (n = 5) were treated with TS or saline 5 min prior to fentanyl administration. TS [2 mg/kg, intravenous (i.v.)] dose-dependently attenuated fentanyl-induced respiratory depression versus saline + fentanyl group. Furthermore, pre-treatment with TS (2 mg/kg, i.v.) increased arterial oxygen saturation to 76.5 ± 2.0% at 5 min after fentanyl injection, compared with 35.9 ± 2.5% in saline pre-treated rats (P < 0.001), whereas the time to induction of LORR (P > 0.99) and duration of LORR (P = 0.95) did not differ between the “TS + fentanyl” and “saline + fentanyl” group. The antinociceptive effect of fentanyl was not affected by the administration of TS (P = 0.99) in mice (n = 10). In conclusion, we found that TS, a novel non-benzodiazepine anxiolytic/antidepressant drug, could attenuate severe fentanyl-induced respiratory depression and did not affect the analgesic/sedative effect of fentanyl. The clinical application of TS could significantly improve pain management.

Desformylflustrabromine (dFBr), a positive allosteric modulator of the α4β2 nicotinic receptor modulates the hypnotic response to ethanol.

Binge drinking can increase an individual's risk of developing alcohol use disorder (AUD). Ethanol targets multiple neurotransmitter systems; however, not much is known about its effects on the cholinergic system. Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels, the heteromeric α4β2 nAChR being a commonly expressed subtype. Desformylflustrabromine (dFBr), a positive allosteric modulator (PAM), increases the efficacy of α4β2 nAChR invitro and has previously been shown to have translational potential. In this study, we investigated whether dFBr modulates the hypnotic response to ethanol. Ethanol-induced loss of righting reflex (LORR) duration was measured in the presence and absence of dFBr. The β2 nAChR selective antagonist dihydro-β-erythroidine (DHβE) was used to study the involvement of the β2 subunit. Additionally, we used a crosslinking-based western blot assay to estimate changes in total versus intracellular α4 nAChR protein in thalamic tissue of rats treated with vehicle, dFBr, ethanol, or ethanol and dFBr. Lastly, using Xenopus oocyte two-electrode voltage clamp (TEVC) studies, we determined the effects of ethanol and dFBr on α4β2 nAChR. Pretreatment with 6mg/kg dFBr reduced ethanol-induced LORR duration as compared to rats treated with ethanol alone. LORR studies with DHβE suggest that dFBr reduced ethanol-induced LORR duration via the β2 nAChR subunit. Crosslinking-based western analyses revealed that ethanol caused early increases in total and presumably surface thalamic α4 nAChR subunit protein levels. This ethanol-induced α4 nAChR upregulation was significantly reduced in rats pretreated with 6mg/kg dFBr. In TEVC studies, ethanol potentiated ACh-induced currents in α4β2 nAChR, while it slightly reduced dFBr potentiation of maximal ACh currents. Our results suggest that thalamic nAChRs containing the α4 subunit are rapidly upregulated by a single intoxicating dose of ethanol. Furthermore, dFBr, an α4β2 nAChR-selective PAM, significantly attenuates the hypnotic response to ethanol via actions on β2 nAChR. Overall, these results indicate that dFBr represents an option to reverse ethanol intoxication.

GluN2A-selective positive allosteric modulator-nalmefene-flumazenil reverses ketamine-fentanyl-dexmedetomidine-induced anesthesia and analgesia in rats

Anesthetics are used to produce hypnosis and analgesic effects during surgery, but anesthesia for a long time after the operation is not conducive to the recovery of animals or patients. Therefore, finding appropriate treatments to counter the effects of anesthetics could enhance postoperative recovery. In the current study, we discovered the novel role of a GluN2A-selective positive allosteric modulator (PAM) in ketamine-induced anesthesia and investigated the effects of the PAM combined with nalmefene and flumazenil (PNF) in reversing the actions of an anesthetic combination (ketamine-fentanyl-dexmedetomidine, KFD). PAM treatment dose-dependently decreased the duration of the ketamine-induced loss of righting reflex (LORR). Compared with those in the KFD group, the duration of LORR and the analgesic effect of the KFD + PNF group were obviously decreased. Meanwhile, successive administration of PNF and KFD had no adverse effects on the cardiovascular and respiratory systems. Both the KFD group and the KFD + PNF group showed no changes in hepatic and renal function or cognitive function in rats. Moreover, the recovery of motor coordination of the KFD + PNF group was faster than that of the KFD group. In summary, our results suggest the potential application of the PNF combination as an antagonistic treatment strategy for anesthesia.

Effects of Xingnaojing Injection on Adenosinergic Transmission and Orexin Signaling in Lateral Hypothalamus of Ethanol-Induced Coma Rats.

Acute alcohol exposure induces unconscious condition such as coma whose main physical manifestation is the loss of righting reflex (LORR). Xingnaojing Injection (XNJI), which came from Chinese classic formula An Gong Niu Huang Pill, is widely used for consciousness disorders in China, such as coma. Although XNJI efficiently shortened the duration of LORR induced by acute ethanol, it remains unknown how XNJI acts on ethanol-induced coma (EIC). We performed experiments to examine the effects of XNJI on orexin and adenosine (AD) signaling in the lateral hypothalamic area (LHA) in EIC rats. Results showed that XNJI reduced the duration of LORR, which implied that XNJI promotes recovery form coma. Microdialysis data indicated that acute ethanol significantly increased AD release in the LHA but had no effect on orexin A levels. The qPCR results displayed a significant reduction in the Orexin-1 receptors (OX1R) expression with a concomitant increase in the A1 receptor (A1R) and equilibrative nucleoside transporter type 1 (ENT1) expression in EIC rats. In contrast, XNJI reduced the extracellular AD levels but orexin A levels remained unaffected. XNJI also counteracted the downregulation of the OX1R expression and upregulation of A1R and ENT1 expression caused by EIC. As for ADK expression, XNJI but not ethanol, displayed an upregulation in the LHA in EIC rats. Based on these results, we suggest that XNJI promotes arousal by inhibiting adenosine neurotransmission via reducing AD level and the expression of A1R and ENT1.

Protective effects of probiotics on acute alcohol-induced liver injury in mice through alcohol metabolizing enzymes activation and hepatic TNF-α response reduction

Abstract Purpose The role of tumor necrosis factor-α (TNFα) and toll-like receptor 4 (TLR 4) in the pathogenesis of alcoholic liver disease (ALD) has been widely entrenched. We investigated the profitable effects of Bifidobacterium bifidum YRT3115 and Bifidobacterium animalis subsp. lactis KV9 on tumor necrosis factor-α (TNFα) expression and injury response to acute alcohol gavage in mice. Methods Ninety-six C57BL/6 mice were classified into 4 groups: control, intragastric administration of white spirit, intragastric administration of white spirit + YRT3115 and intragastric administration of white spirit + KV9. The mice were treated daily with probiotics and white spirit until 15 days. The body weight, food intake, liver weight and liver index of mice were measured. Duration of alcohol-induced loss of righting reflex (LORR) was also evaluated. The concentration of ethanol (EtOH) and acetaldehyde (AcH) in mice blood were performed by a head-space GC. The activity of aspartate transaminase (AST) and alanine transaminase (ALT) in mice serum, activity of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in mice liver were performed by commercial kits. The liver mRNA levels of TNF-α, CYP2E1 and TLR 4 were evaluated by RT-PCR. The protein level of TNF-α was evaluated by Western-blot. Results After intragastric administration white spirit, B. animalis subsp. lactis KV9 reduced duration of LORR, recovery of loss of righting reflex (RORR) and was significantly reduced the concentration of EtOH in blood of mice, by 15.69% at 30 min, 15.01% at 60 min and 45.01% at 120 min (p Conclusion This paper demonstrated that B. animalis subsp. lactis KV9 supplementation reduced markedly hepatic inflammation, liver injury and improve alcohol metabolism, but B. bifidum YRT3115 was not. These results suggested firmly that treatment with B. animalis subsp. lactis KV9 could potential ameliorate liver from the injury of alcohol.

Influence of lipopolysaccharide on diazepam-modified loss of righting reflex duration by pentobarbital treatment in mice

Benzodiazepine receptor agonists are widely prescribed therapeutic agents, alter gamma-aminobutyric acid (GABA)A receptor function, and have hypnotic, anxiolytic, anticonvulsant, and antispastic effects. GABAA receptor activity increases under systemic inflammatory conditions. We investigated the effect of benzodiazepine receptor agonists on pentobarbital-induced loss of righting reflex (LORR) duration using a mouse model of lipopolysaccharide (LPS)-induced inflammation. We assessed pentobarbital-induced LORR duration 24 h after LPS treatment in mice. Additionally, we examined the microglial response by immunohistochemistry and serum IL-6 and TNF-α concentrations in mice. LPS treatment significantly increased the duration of pentobarbital-induced LORR in mice treated with benzodiazepine receptor agonists (diazepam and brotizolam) and a GABAA receptor agonist (muscimol) compared to that of mice treated with vehicle. These effects were blocked by bicuculline, a GABAA receptor antagonist. LPS significantly increased the number of ionized calcium binding adapter molecule-1-positive hippocampal cells 2 and 24 h after treatment. The enhancing effect of diazepam in LPS-treated mice was significantly reduced by minocycline. These findings suggest that LPS enhances pentobarbital-induced LORR duration in mice treated with benzodiazepine via GABAA receptor activity.

Late aging alters behavioral sensitivity to ethanol in a sex-specific manner in Fischer 344 rats

Responsiveness to ethanol (EtOH) differs as a function of age. Adolescent rodents are less sensitive than adults to the sedative effects of EtOH, whereas they show enhanced sensitivity to EtOH-induced social facilitation. Late aging is associated with a natural decline in social behavior and aging-related peculiarities in sensitivity to EtOH have been largely unexplored. Whether there are sex differences in the behavioral response to EtOH during late aging remains unknown. Thus, behavioral responses to EtOH in male and female Fischer (F) 344 rats aged 4–5 months (adult) and 19–20 months (aging) were examined. First, the effects of saline and EtOH (0.5 and 0.75 g/kg) on social interaction were assessed. Social investigation and contact behavior were lower in aging animals and higher in females. Interestingly, in aged females, social contact behavior was increased following a 0.5 g/kg EtOH dose, whereas the same dose suppressed social contact in aged males. Behavioral sensitivity to the sedative effects of 3.0 and 3.5 g/kg EtOH was assessed with the loss of righting reflex (LORR) test. Although latency to LORR did not differ as a function of age or sex, aged rats showed significantly greater LORR duration and significantly lower blood ethanol concentrations (BECs) at regaining of the righting reflex relative to adults. In addition, females had a lower LORR duration, regardless of age; no sex differences were evident in BECs at awakening. In a second experiment, blood ethanol concentrations (BECs) over time were assessed following 0.75, 1.5, and 3.0 g/kg EtOH in 3-, 12-, and 18-month-old male and female F344 rats. Aged rats had higher peak BECs following 3.0 g/kg EtOH, whereas few age or sex differences were apparent at lower doses. Taken together, these data indicate that late aging is associated with altered sensitivity to the social facilitating effects and sedative effects of EtOH.

Apremilast Alters Behavioral Responses to Ethanol in Mice: II. Increased Sedation, Intoxication, and Reduced Acute Functional Tolerance.

In our companion paper, we reported that the phosphodiesterase type 4 inhibitor apremilast reduced ethanol (EtOH) intake and preference in different drinking models in male and female C57BL/6J mice. In this study, we measured the effects of apremilast on other behaviors that are correlated with EtOH consumption. The effects of apremilast (20mg/kg) on the following behaviors were studied in male and female C57BL/6J mice: locomotor response to a novel situation; EtOH- and lithium chloride (LiCl)-induced conditioned taste aversion (CTA) to saccharin; conditioned place preference (CPP) and conditioned place avoidance (CPA) to EtOH; severity of handling-induced convulsions after EtOH administration; EtOH-induced anxiolytic-like behavior in the elevated plus maze; duration of EtOH-induced loss of righting reflex (LORR); recovery from EtOH-induced motor impairment on the rotarod; and acute functional tolerance (AFT) to EtOH's ataxic effects. Apremilast did not change the acquisition of EtOH-induced CPP, severity of acute withdrawal from EtOH, or EtOH's anxiolytic-like effect. Apremilast did not alter the extinction of EtOH- or LiCl-induced CTA, but may interfere with acquisition of CTA to EtOH. Apremilast increased the acquisition of CPA to EtOH, reduced locomotor responses to a novel situation, and prolonged the duration of LORR and the recovery from acute motor incoordination induced by EtOH. The longer recovery from the ataxic effect may be attributed to reduced development of AFT to EtOH. Our results suggest that apremilast increases the duration of EtOH intoxication by reducing AFT. Apremilast also reduces some aspects of general reward and increases EtOH's aversive properties, which might also contribute to its ability to reduce EtOH drinking.

Behavioral Characterization of β-Arrestin 1 Knockout Mice in Anxiety-Like and Alcohol Behaviors.

β-Arrestin 1 and 2 are highly expressed proteins involved in the desensitization of G protein-coupled receptor signaling which also regulate a variety of intracellular signaling pathways. Gene knockout (KO) studies suggest that the two isoforms are not homologous in their effects on baseline and drug-induced behavior; yet, the role of β-arrestin 1 in the central nervous system has been less investigated compared to β-arrestin 2. Here, we investigate how global β-arrestin 1 KO affects anxiety-like and alcohol-related behaviors in male and female C57BL/6 mice. We observed increased baseline locomotor activity in β-arrestin 1 KO animals compared with wild-type (WT) or heterozygous (HET) mice with a sex effect. KO male mice were less anxious in a light/dark transition test, although this effect may have been confounded by increased locomotor activity. No differences in sucrose intake were observed between genotypes or sexes. Female β-arrestin 1 KO mice consumed more 10% alcohol than HET females in a limited 4-h access, two-bottle choice, drinking-in-the-dark model. In a 20% alcohol binge-like access model, female KO animals consumed significantly more alcohol than HET and WT females. A significant sex effect was observed in both alcohol consumption models, with female mice consuming greater amounts of alcohol than males relative to body weight. Increased sensitivity to latency to loss of righting reflex (LORR) was observed in β-arrestin 1 KO mice although no differences were observed in duration of LORR. Overall, our efforts suggest that β-arrestin 1 may be protective against increased alcohol consumption in females and hyperactivity in both sexes.

Altered Function and Expression of ABC Transporters at the Blood–Brain Barrier and Increased Brain Distribution of Phenobarbital in Acute Liver Failure Mice

This study investigated alterations in the function and expression of P-glycoprotein (P-GP), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 2 (MRP2) at the blood–brain barrier (BBB) of acute liver failure (ALF) mice and its clinical significance. ALF mice were developed using intraperitoneal injection of thioacetamide. P-GP, BCRP, and MRP2 functions were determined by measuring the ratios of brain-to-plasma concentration of rhodamine 123, prazosin, and dinitrophenyl-S-glutathione, respectively. The mRNA and proteins expression levels of P-GP, BCRP, and MRP2 were evaluated with quantitative real-time PCR and western blot, respectively. MDCK-MDR1 and HCMEC/D3 cells were used to document the effects of the abnormally altered components in serum of ALF mice on the function and expression of P-GP. The clinical significance of alteration in P-GP function and expression was investigated by determining the distribution of the P-GP substrate phenobarbital (60 mg/kg, intravenous administration) in the brain and loss of righting reflex (LORR) induced by the drug (100 mg/kg). The results showed that ALF significantly downregulated the function and expression of both P-GP and BCRP, but increased the function and expression of MRP2 in the brain of mice. Cell study showed that increased chenodeoxycholic acid may be a reason behind the downregulated P-GP function and expression. Compared with control mice, ALF mice showed a significantly higher brain concentration of phenobarbital and higher brain-to-plasma concentration ratios. In accordance, ALF mice showed a significantly larger duration of LORR and shorter latency time of LORR by phenobarbital, inferring the enhanced pharmacological effect of phenobarbital on the central nervous system (CNS). In conclusion, the function and expression of P-GP and BCRP decreased, while the function and expression of MRP2 increased in the brain of ALF mice. The attenuated function and expression of P-GP at the BBB might enhance phenobarbital distribution in the brain and increase phenobarbital efficacy on the CNS of ALF mice.

General anesthetic actions on GABAA receptors in vivo are reduced in phospholipase C-related catalytically inactive protein knockout mice.

The aim of this study was to investigate the action of general anesthetics in phospholipase C-related catalytically inactive protein (PRIP)-knockout (KO) mice that alter GABAA receptor signaling. PRIP regulates the intracellular trafficking of β subunit-containing GABAA receptors in vitro. In this study, we examined the effects of intravenous anesthetics, propofol and etomidate that act via β subunit-containing GABAA receptors, in wild-type and Prip-KO mice. Mice were intraperitoneally injected with a drug, and a loss of righting reflex (LORR) assay and an electroencephalogram analysis were performed. The cell surface expression of GABAA receptor β3 subunit detected by immunoblotting was decreased in Prip-knockout brain compared with that in wild-type brain without changing the expression of other GABAA receptor subunits. Propofol-treated Prip-KO mice exhibited significantly shorter duration of LORR and had lower total anesthetic score than wild-type mice in the LORR assay. The average duration of sleep time in an electroencephalogram analysis was shorter in propofol-treated Prip-KO mice than in wild-type mice. The hypnotic action of etomidate was also reduced in Prip-KO mice. However, ketamine, an NMDA receptor antagonist, had similar effects in the two genotypes. PRIP regulates the cell surface expression of the GABAA receptor β3 subunit and modulates general anesthetic action in vivo. Elucidation of the involved regulatory mechanisms of GABAA receptor-dependent signaling would inform the development of safer anesthetic therapies for clinical applications.

Transgenerational Transmission of the Effect of Gestational Ethanol Exposure on Ethanol Use-Related Behavior.

Prenatal alcohol exposure (PAE) enhances the risk for alcoholism by increasing the propensity to consume alcohol and altering neurophysiological response to alcohol challenge. Trans-generationally transmittable genetic alterations have been implicated in these behavioral changes. To date, transgenerational transmission of PAE-induced behavioral responses to alcohol has never been experimentally investigated. Therefore, we explored the transgenerational transmission of PAE-induced behavioral effects across 3 generations. Pregnant Sprague Dawley dams received 1g/kg ethanol (EtOH) or water daily on gestational days 17 through 20 via gavage, or remained untreated in their home cages. To produce second filial (F2) or F3 generations, similarly treated adult F1 or F2 offspring were mated and left undisturbed through gestation. On postnatal day (PND) 14, male and female F1, F2, and F3 offspring were tested for consumption of 5% (w/v) EtOH (in water), or water. Using the loss of righting reflex (LORR) paradigm on PND 42, F1 and F2 adolescent male offspring were tested for sensitivity to acute EtOH-induced sedation-hypnosis at 3.5 or 4.5g/kg dose. F3 male adolescents were similarly tested at 3.5g/kg dose. Blood EtOH concentration (BEC) was measured at waking. EtOH exposure increased EtOH consumption compared to both water and untreated control groups in all generations. EtOH-treated group F1 and F2 adolescents displayed attenuated LORR duration compared to the water group. No attenuated LORR was observed in the F3 generation. BEC at waking corroborated with the significant LORR duration differences while also revealing differences between untreated control and water groups in F1 and F2 generations. Our results provide novel behavioral evidence attesting that late gestational moderate EtOH exposure increases EtOH intake across 3 generations and may alter sensitivity to EtOH-induced sedation-hypnosis across 2 generations.

The cannabinoid receptor 2 agonist β-caryophyllene reduces voluntary alcohol intake and attenuated ethanol-induced place preference and sensitivity in mice

Introduction: Several recent studies have suggested that brain cannabinoid receptor 2 (CB2) plays a major role in alcohol reward. In fact, the implication of cannabinoid neurotransmission in the reinforcing effects of ethanol (EtOH) is becoming increasingly evident. Objectives: The CB2 receptor agonist β -caryophyllene (BCP) was used to investigate the role of the CB2 receptors in mediating alcohol intake and ethanol-induced conditioned place preference (EtOH-CPP) and sensitivity in mice. Material and methods: The effect of BCP on alcohol intake was evaluated using the standard two-bottle-choice drinking method. The mice were presented with increasing EtOH concentrations and its consumption was measured daily. Consumption of saccharin and quinine solutions were measured following the EtOH preference tests. Finally, the effect of BCP on alcohol reward and sensitivity was tested using an unbiased EtOH-CPP and loss of righting reflex (LORR) procedures, respectively. Results: BCP dose-dependently decreased alcohol consumption and preference. In addition, BCP-injected mice did not show any difference from vehicle mice in total fluid intake in a 24-hour paradigm or in their intake of graded concentrations of saccharin or quinine, suggesting that the CB2 receptor activation did not alter taste function. More importantly, BCP inhibited EtOH-CPP acquisition and exacerbated LORR duration. Interestingly, these effects were abrogated when mice were preinjected with a selective CB2 receptor antagonist, AM630. Conclusions: The CB2 receptor system appears to be involved in alcohol dependence and sensitivity and may represent a potential pharmacological target for the treatment of alcoholism. Acknowledgements: Dr Amine Bahi for supervising this project.

The cannabinoid receptor 2 agonist, β-caryophyllene, reduced voluntary alcohol intake and attenuated ethanol-induced place preference and sensitivity in mice

Several recent studies have suggested that brain CB2 cannabinoid receptors play a major role in alcohol reward. In fact, the implication of cannabinoid neurotransmission in the reinforcing effects of ethanol (EtOH) is becoming increasingly evident. The CB2 receptor agonist, β-caryophyllene (BCP) was used to investigate the role of the CB2 receptors in mediating alcohol intake and ethanol-induced conditioned place preference (EtOH-CPP) and sensitivity in mice. The effect of BCP on alcohol intake was evaluated using the standard two-bottle choice drinking method. The mice were presented with increasing EtOH concentrations and its consumption was measured daily. Consumption of saccharin and quinine solutions was measured following the EtOH preference tests. Finally, the effect of BCP on alcohol reward and sensitivity was tested using an unbiased EtOH-CPP and loss of righting-reflex (LORR) procedures, respectively. BCP dose-dependently decreased alcohol consumption and preference. Additionally, BCP-injected mice did not show any difference from vehicle mice in total fluid intake in a 24-hour paradigm nor in their intake of graded concentrations of saccharin or quinine, suggesting that the CB2 receptor activation did not alter taste function. More importantly, BCP inhibited EtOH-CPP acquisition and exacerbated LORR duration. Interestingly, these effects were abrogated when mice were pre-injected with a selective CB2 receptor antagonist, AM630. Overall, the CB2 receptor system appears to be involved in alcohol dependence and sensitivity and may represent a potential pharmacological target for the treatment of alcoholism.

Nalmefene reverses carfentanil-induced loss of righting reflex and respiratory depression in rats

Reversing the respiratory depression induced by carfentanil involves intravenous administration of naloxone or naltrexone, but this treatment has disadvantages. Hence, finding a more appropriate treatment to counter the depressive actions of carfentanil is needed. In the present study, with the naloxone as a control, we investigated the efficacy of nalmefene for countering the depressive actions of carfentanil. Rats were treated successively with carfentanil (10μg/kg, i.v.) and nalmefene (9.4–150.0μg/kg, i.m.), and the duration of loss of righting reflex (LORR) recorded. Respiratory parameters were measured in free-moving rats using a whole-body plethysmograph after rats were administered carfentanil (20μg/kg, i.v.) and nalmefene (9.4–150.0μg/kg, i.m.) sequentially. The parameters of arterial blood gases were also examined. Nalmefene (9.4–150.0μg/kg, i.m.) treatment dose-dependently decreased the duration of carfentanil-induced LORR. The respiratory rate after 60min of nalmefene (150.0μg/kg, i.m.) treatment increased from 34.3±5.3bursts/min to 117.8±18.9bursts/min, and enhanced pause decreased from 1.1±0.1 to 0.4±0.1, and was close to those of normal rats. Furthermore, nalmefene (37.5–150.0μg/kg) treatment could enable the PaO2, SaO2 and PaCO2 to approach normal levels 10min (15min after carfentanil injection) or 30min (25min after carfentanil injection) after injection. While, a single injection of naloxone (150.0μg/kg, i.m.) only achieved partial remission of respiratory depression. These data suggest that nalmefene more effectively counters the depressive actions induced by carfentanil and is a more appropriate treatment to antagonize carfentanil toxicity compared with naloxone.