[Dexmedetomidine improves alcohol withdrawal symptom via activating α2 adrenergic receptor in rat hippocampus].

The purpose of this study was to investigate the effects of α2 adrenergic receptor agonist dexmedetomidine on withdrawal symptoms in alcohol-dependent rats and the underlying mechanism, so as to provide a scientific basis for the treatment of alcohol withdrawal syndrome (AWS). Adult Sprague-Dawley (SD) male rats were orally administered with 6% aqueous alcohol continuously for 28 d to establish alcohol drinking model, and then stopped drinking to induce AWS. Enzyme-linked immunosorbent assay (ELISA) was used to determine the content of norepinephrine (NE) in the locus coeruleus and hippocampus of rats. Dexmedetomidine (5, 10, and 20 µg/kg) was intraperitoneally injected respectively when the rats showed significant AWS. In some rats, α2 adrenergic receptor antagonist yohimbine was injected into hippocampus in advance. The results showed that, compared with the control group, the 6 h withdrawal group exhibited significantly increased AWS score and amount of repeat drinking. The NE contents in hippocampus and locus coeruleus of the last drinking and the 6 h withdrawal groups were significantly increased compared with those of the control group. Dexmedetomidine intervention significantly decreased AWS score and hippocampus NE content in the 6 h withdrawal group, while yohimbine could reverse these effects of dexmedetomidine. These results suggest that dexmedetomidine might improve the withdrawal symptoms in alcohol-dependent rats via activating α2 adrenergic receptor.

Nitric Oxide Signaling Pathway in Ventral Tegmental Area is Involved in Regulation of 7,8-Dihydroxyflavone on Alcohol Consumption in Rats.

We recently reported that intraperitoneal injection of 7,8-dihydroxyflavone (7,8-DHF), a brain-derived neurotrophic factor-mimicking small compound, could attenuate alcohol-related behaviors in a two-bottle choice ethanol consumption procedure (IA2BC) in rats via tropomyosin receptor kinase B in the ventral tegmental area (VTA), which is closely related to alcohol use disorder. However, the detailed mechanisms underlying the regulation of 7,8-DHF on alcohol drinking behavior remain elusive. In this study, we determined the role of nitric oxide (NO), a pleiotropic signaling molecule, in the VTA in the action of 7,8-DHF upon alcohol drinking behavior. Intermittent alcohol exposure led to the overexpression of NO in the VTA, especially 72 h after withdrawal from four weeks of ethanol exposure in IA2BC rats. A higher amount of alcohol intake was also found at the same time point, consistent with the overexpression of NO in the VTA. Microinjection of NG-Nitro-l-Arginine Methyl Ester, (NO synthase inhibitor) or 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (NO scavenger) into the VTA inhibited alcohol intake, whereas application of S-Nitroso-N-acetyl-DL-penicillamine (SNAP, the NO donor) in the VTA further enhanced alcohol consumption in IA2BC rats. Interestingly, either 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (a sGC inhibitor) or KT5823 [a selective protein kinase G (PKG) inhibitor] blocked NO's enhancing effect on ethanol intake. Intraperitoneal injection of 7,8-DHF reduced the overexpression of NO; SNAP microinjected into the VTA reversed the inhibitory effects of 7,8-DHF on alcohol consumption. Our findings suggest that NO-cGMP-PKG might be involved in regulation of 7,8-DHF on alcohol consumption in IA2BC rats.

Regulatory role of miR-129 and miR-384-5p on apoptosis induced by oxygen and glucose deprivation in PC12 cell.

This study aimed to establish the role of miR-129 and miR-384-5p in cerebral ischemia-induced apoptosis. Using PC12 cells transfected with miR-129 or miR-384-5p mimics or inhibitors, oxygen glucose deprivation (OGD) conditions were applied for 4 h to simulate transient cerebral ischemia. Apoptotic phenotypes were assessed via lactate dehydrogenase (LDH) assay, MTT cell metabolism assay, and fluorescence-activated cell sorting (FACS). The effect of miR overexpression and inhibition was evaluated by protein and mRNA detection of bcl-2 and caspase-3, critical apoptosis factors. Finally, the direct relationship of miR-129 and bcl-2 and miR-384-5p and caspase-3 was measured by luciferase reporter assay. The overexpression of miR-384-5p and miR-129 deficiency significantly enhanced cell viability, reduced LDH release, and inhibited apoptosis. By contrast, overexpression of miR-129 and miR-384-5p deficiency aggravated hypoxia-induced apoptosis and cell injury. miR-129 overexpression significantly reduced mRNA and protein levels of bcl-2 and miR-129 inhibition significantly increased mRNA and protein levels of bcl-2 in hypoxic cells.miR-384-5p overexpression significantly reduced protein levels of caspase-3 while miR-384-5p deficiency significantly increased protein levels of caspase-3. However, no changes were observed in caspase-3 mRNA in either transfection paradigm. Finally, luciferase reporter assay confirmed caspase-3 to be a direct target of miR-384-5p; however, no binding activity was detected between bcl-2 and miR-129.Transient cerebral ischemia induces differential expression of miR-129 and miR-384-5p which influences apoptosis by regulating apoptotic factors caspase-3 and bcl-2, thereby participating in the pathological mechanism of cerebral ischemia, and becoming potential targets for the treatment of ischemic cerebral injury in the future.

Validation and Factor Analysis of the Obsessive Compulsive Drinking Scale (OCDS) in the Chinese Population.

Obsessive Compulsive Drinking Scale (OCDS) was established and introduced to measure the craving for alcohol and the severity of alcohol dependence. However, the Chinese version of OCDS is still unavailable and has not been validated in the Chinese population. We tended to translate and validate the OCDS in Chinese. We translated original OCDS into Chinese through bi-direction translations and tested the reliability and validity. We found that Chinese OCDS had high internal consistency and good test-retest reliability. The Chinese OCDS also presented good internal structure to reflect the severity of alcohol dependence. The Chinese OCDS could be used in clinical studies and research among the Chinese population.

7,8-Dihydroxyflavone Alleviates Anxiety-Like Behavior Induced by Chronic Alcohol Exposure in Mice Involving Tropomyosin-Related Kinase B in the Amygdala.

Alcohol use-associated disorders are highly comorbid with anxiety disorders; however, their mechanism remains unknown. The amygdala plays a central role in anxiety. We recently found that 7,8-dihydroxyflavone (7,8-DHF) significantly reduces withdrawal symptoms in a rat model of chronic intermittent alcohol (ethanol) exposure. This study aimed to determine the role of 7,8-DHF in regulating anxiety induced by chronic alcohol exposure and its associated underlying mechanism. Male C57BL/6J mice were exposed to chronic intermittent alcohol for 3 weeks followed by alcohol withdrawal for 12 h with or without 7,8-DHF administered intraperitoneally. All mice were tested using an open field test and elevated plus maze to assess anxiety-like behaviors. Synaptic activity and intrinsic excitability in basal and lateral amygdala (BLA) neurons were assessed using electrophysiological recordings. 7,8-DHF alleviated alcohol-induced anxiety-like behavior and attenuated alcohol-induced enhancement of activities in BLA pyramidal neurons. Furthermore, 7,8-DHF prevented alcohol withdrawal-evoked augmentation of glutamatergic transmission in the amygdala and had no effect on GABAergic transmission in the amygdala, as demonstrated by unaltered frequency and amplitude of spontaneous inhibitory postsynaptic currents. Microinjection of K252a, a tropomyosin-related kinase B (TrkB) antagonist, into the BLA blocked the effects of 7,8-DHF on anxiety-like behavior and neuronal activity in the BLA. Our findings suggest that 7,8-DHF alleviates alcohol-induced anxiety-like behavior induced by chronic alcohol exposure through regulation of glutamate transmission involving TrKB in the BLA.

7,8-Dihydroxyflavone Attenuates Alcohol-Related Behavior in Rat Models of Alcohol Consumption via TrkB in the Ventral Tegmental Area.

Alcohol use disorder (AUD) is a ubiquitous substance use disorder in the world, of which neural mechanisms remain unclear. Alcohol consumption induces neuro-adaptations in the dopaminergic system originating from the ventral tegmental area (VTA), an important brain region for the reward function in AUD. Endogenous brain-derived neurotrophic factor (BDNF)-TrkB implicated in the development of neuroplasticity, including long-term potentiation of GABAergic synapses (LTP GABA ). We previously found that ethanol blocks LTP GABA in the VTA, either in vivo or in vitro. 7,8-dihydroflavone (7,8-DHF), a BDNF-mimicking small compound, was recently found to penetrate the blood-brain barrier to mimic the biological role of BDNF-TrkB. In this study, we demonstrate that repeated ethanol consumption (including intermittent and continuous ethanol exposure) results in low expression of BDNF in rat VTA. The amount of ethanol intake enhances significantly in rats with intermittent ethanol exposure after 72 h abstinence. Withdrawal signs emerge in rats with continuous ethanol exposure within 3 days after abstinence. Using behavioral tests, intraperitoneal injection of 7,8-DHF can reduce excessive ethanol consumption and preference as well as withdrawal signs in rats with repeated ethanol exposure. Interestingly, microinjection of K252a, an antagonist of TrkB, into the VTA blocks the effects of 7,8-DHF on ethanol-related behaviors. Furthermore, direct microinjection of BDNF into the VTA mimics the effect of 7,8-DHF on ethanol related behaviors. Taken together, 7,8-DHF attenuates alcohol-related behaviors in rats undergoing alcohol consumption via TrkB in the VTA. Our findings suggest BDNF-TrkB in VTA is a part of regulating signals for opposing neural adaptations in AUD, and 7,8-DHF may serve as a potential candidate for treating alcoholism.

Conformational activation of ribosome recycling by intra- and inter-molecular dynamics of RRF.

Ribosome recycling is the final step of the cyclic process of translation, where the post-termination complex (PoTC) is disassembled by the concerted action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in the sub-second time range. Since, however, both the RRF and PoTC display highly dynamic action during this process, it is difficult to assess the molecular details of the interactions between the factors and the ribosome that are essential for rapid subunit separation. Here we characterized the molecular dynamics of RRF and PoTC by combined use of molecular dynamics simulations, single molecule fluorescence detection and single-particle cryo-EM analysis, with time resolutions in the sub-millisecond to minute range. We found that RRF displays two-layer dynamics: intra- and inter-molecular dynamics during ribosome splitting. The intra-molecular dynamics exhibits two different configurations of RRF: 'bent' and 'extended'. A single-site mutant of RRF increases its propensity to the 'extended' conformation and leads to a higher binding affinity of RRF to the PoTC. The inter-molecular dynamics between RRF and EF-G in the PoTC reveals that the domain IV of EF-G pushes against the domain II of RRF, triggering the disruption of the major inter-subunit bridge B2a, and catalyzes the splitting.

Nicotine inhibits microglial proliferation and is neuroprotective in global ischemia rats.

Ischemic injury in rodent models reliably leads to the activation of microglia, which might play a detrimental role in neuronal survival. Our preliminary studies suggest that nicotine plays a potential role in decreasing the numbers of cultured microglia in vitro. In the present study, we found treatment with nicotine 2, 6, and 12 h after ischemia for 7 days significantly increased the survival of CA1 pyramidal neurons in ischemia/reperfusion rats. This effect was accompanied by a significant reduction in the increase of microglia rather than astrocytes, as well as a significant reduction of enhanced expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1ß) in CA1 induced by ischemia/reperfusion. Nicotine inhibits microglial proliferation in primary cultures with and without the stimulation of granulocyte-macrophage colony-stimulating factor (GM-CSF). Pre-treatment with α-bungarotoxin, a selective α7 nicotinic acetylcholine receptor (α7 nAChR) antagonist, could prevent the inhibitory effects of nicotine on cultured microglial proliferation suggesting that nicotine inhibits the microglial proliferation in an α7 nAChR-dependent fashion. Our results suggest that nicotine inhibits the inflammation mediated by microglia via α7 nAChR and is neuroprotective against ischemic stroke, even when administered 12 h after the insult. α7 nAChR agonists may have uses as anti-ischemic compounds in humans.

Feline bone marrow-derived mesenchymal stromal cells (MSCs) show similar phenotype and functions with regards to neuronal differentiation as human MSCs.

Mesenchymal stromal cells (MSCs) show promise for treatment of a variety of neurological and other disorders. Cat has a high degree of linkage with the human genome and has been used as a model for analysis of neurological disorders such as stroke, Alzheimer's disease and motor disorders. The present study was designed to characterize bone marrow-derived MSCs from cats and to investigate the capacity to generate functional peptidergic neurons. MSCs were expanded with cells from the femurs of cats and then characterized by phenotype and function. Phenotypically, feline and human MSCs shared surface markers, and lacked hematopoietic markers, with similar morphology. As compared to a subset of human MSCs, feline MSCs showed no evidence of the major histocompatibility class II. Since the literature suggested Stro-1 as an indicator of pluripotency, we compared early and late passages feline MSCs and found its expression in >90% of the cells. However, the early passage cells showed two distinct populations of Stro-1-expressing cells. At passage 5, the MSCs were more homogeneous with regards to Stro-1 expression. The passage 5 MSCs differentiated to osteogenic and adipogenic cells, and generated neurons with electrophysiological properties. This correlated with the expression of mature neuronal markers with concomitant decrease in stem cell-associated genes. At day 12 induction, the cells were positive for MAP2, Neuronal Nuclei, tubulin ßIII, Tau and synaptophysin. This correlated with electrophysiological maturity as presented by excitatory postsynaptic potentials (EPSPs). The findings indicate that the cat may constitute a promising biomedical model for evaluation of novel therapies such as stem cell therapy in such neurological disorders as Alzheimer's disease and stroke.

[Involvement of inhibition of nucleus GAPDH over-expression in erythropoietin's reduction of neuronal apoptosis induced by brain ischemia/reperfusion in rats].

To study whether recombinant human erythropoietin (rhEPO) reduces neuronal apoptosis through inhibiting over-expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in nucleus induced by brain ischemia/reperfusion in rats, 48 adult Sprague-Dawley rats were randomly divided into 3 groups: sham, saline and EPO groups. Animal models of brain ischemia/reperfusion were established by middle cerebral artery occlusion in rats. The effects of EPO on the sizes of ischemia tissue were observed by TTC staining. The over-expression of GAPDH in nucleus was detected by Hoechst-33258 and anti-GAPDH antibody double staining. The neuronal apoptosis in penumbral was detected by Nissl's staining and Hoechst-33258 immunofluorescence, respectively. The results showed that rhEPO treatment (3 000 U/kg, three times daily, i.p.) apparently reduced the sizes of infarct brain tissue in ischemia/reperfusion rats. rhEPO inhibited over-expression of GAPDH in nucleus of apoptotic neurons. In the meantime rhEPO decreased the number of apoptotic neurons in ischemia/reperfusion rats. These results suggest that rhEPO may induced reduction of neuronal apoptosis in penumbra may be through inhibiting over-expression of GAPDH in nucleus of apoptotic neurons induced by ischemia/reperfusion. Reduction of GAPDH over-expression in nucleus may play a pivotal role in EPO inhibiting neuronal apoptosis in cerebral ischemia/reperfusion rats, providing experimental evidence for EPO neuro-protecting effects against ischemia/reperfusion.

Ethanol blocks long-term potentiation of GABAergic synapses in the ventral tegmental area involving mu-opioid receptors.

It is well documented that ethanol exposure alters GABA (gamma-aminobutyric acid)-releasing synapses, and ethanol addiction is associated with endogenous opioid system. Emerging evidence indicates that opioids block long-term potentiation in the fast inhibitory GABA(A) receptor synapses (LTP(GABA)) onto dopamine-containing neurons in the ventral tegmental area (VTA), a brain region essential for reward-seeking behavior. However, how ethanol affects LTP(GABA) is not known. We report here that in acute midbrain slices from rats, clinically relevant concentrations of ethanol applied both in vitro and in vivo prevents LTP(GABA), which is reversed, respectively, by in vitro and in vivo administration of naloxone, a mu-opioid receptor (MOR) antagonist. Furthermore, the blockade of LTP(GABA) induced by a brief in vitro ethanol treatment is mimicked by DAMGO ([D-Ala(2), N-MePhe(4), Gly-ol]-enkephalin), a MOR agonist. Paired-pulse ratios are similar in slices, 24 h after in vivo injection with either saline or ethanol. Sp-cAMPS, a stable cAMP analog, and pCPT-cGMP, a cGMP analog, potentiates GABA(A)-mediated inhibitory postsynaptic currents in slices from ethanol-treated rats, indicating that a single in vivo ethanol exposure does not maximally increase GABA release, instead, ethanol produces a long-lasting inability to generate LTP(GABA). These neuroadaptations to ethanol might contribute to early stage of addiction.

Propofol facilitates glutamatergic transmission to neurons of the ventrolateral preoptic nucleus.

BACKGROUND: There is much evidence that the sedative component of anesthesia is mediated by gamma-aminobutyric acid type A (GABA(A)) receptors on hypothalamic neurons responsible for arousal, notably in the tuberomammillary nucleus. These GABA(A) receptors are targeted by gamma-aminobutyric acid-mediated (GABAergic) neurons in the ventrolateral preoptic area (VLPO): When these neurons become active, they inhibit the arousal-producing nuclei and induce sleep. According to recent studies, propofol induces sedation by enhancing VLPO-induced synaptic inhibition, making the target cells more responsive to GABA(A). The authors explored the possibility that propofol also promotes sedation less directly by facilitating excitatory inputs to the VLPO GABAergic neurons. METHODS: Spontaneous excitatory postsynaptic currents were recorded from VLPO cells-principally mechanically isolated, but also in slices from rats. RESULTS: In isolated VLPO GABAergic neurons, propofol increased the frequency of glutamatergic spontaneous excitatory postsynaptic currents without affecting their mean amplitude. The action of propofol was mimicked by muscimol and prevented by gabazine, respectively a specific agonist and antagonist at GABA(A) receptors. It was also suppressed by bumetanide, a blocker of Na-K-Cl cotransporter-mediated inward Cl transport. In slices, propofol also increased the frequency of spontaneous excitatory postsynaptic currents and, at low doses, accelerated firing of VLPO cells. CONCLUSION: Propofol induces sedation, at least in part, by increasing firing of GABAergic neurons in the VLPO, indirectly by activation of GABA(A) receptors on glutamatergic afferents: Because these axons/terminals have a relatively high internal Cl concentration, they are depolarized by GABAergic agents such as propofol, which thus enhance glutamate release.

[Phentolamine antagonizes the effects of norepinephrine on the activity of pain-related neurons in the parafascicular nucleus of morphine-dependent rats].

OBJECTIVE: To examine the antagonization of phentolamine against the effects of norepinephrine (NE) on the activity of pain-related neurons in the parafascicular nucleus of morphine-dependent rats. METHODS: Electric impulses were applied as nociceptive stimulus to the right sciatic nerve of morphine-dependent rats, and the discharges of the pain-related neurons in the parafascicular nucleus were recorded by extracellular recording method with glass microelectrodes. RESULTS: Intracerebroventricular injection of norepinephrine resulted in the inhibition of evoked response of the pain-excited neurons as well as the excitation of evoked response of the pain-inhibiting neurons. Both the inhibitory effect on the electric discharges of the pain-excited neurons and the excitatory effect on the pain-inhibiting neurons of norepinephrine were almost completely blocked by intracerebroventricular administration of phentolamine. CONCLUSION: Phentolamine antagonizes the inhibitory effect of norepinephrine on the activity of pain-related neurons in the parafascicular nucleus in morphine-dependent rats, and norepinephrine may play an important role in the integration of the pain signal through the alpha-receptors.