Molecular mechanisms underlying the effects of statins in the central nervous system.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, commonly referred to as statins, are widely used in the treatment of dyslipidaemia, in addition to providing primary and secondary prevention against cardiovascular disease and stroke. Statins' effects on the central nervous system (CNS), particularly on cognition and neurological disorders such as stroke and multiple sclerosis, have received increasing attention in recent years, both within the scientific community and in the media. Current understanding of statins' effects is limited by a lack of mechanism-based studies, as well as the assumption that all statins have the same pharmacological effect in the central nervous system. This review aims to provide an updated discussion on the molecular mechanisms contributing to statins' possible effects on cognitive function, neurodegenerative disease, and various neurological disorders such as stroke, epilepsy, depression and CNS cancers. Additionally, the pharmacokinetic differences between statins and how these may result in statin-specific neurological effects are also discussed.

Reversal of quinpirole inhibition of ventral tegmental area neurons is linked to the phosphatidylinositol system and is induced by agonists linked to G(q).

Putative dopaminergic (pDAergic) ventral tegmental area neurons play an important role in brain pathways related to addiction. Extended exposure of pDAergic neurons to moderate concentrations of dopamine (DA) results in a time-dependent decrease in sensitivity of pDAergic neurons to DA inhibition, a process called dopamine inhibition reversal (DIR). We have shown that DIR is mediated by phospholipase C and conventional protein kinase C through concurrent stimulation of D2 and D1-like receptors. In the present study, we further characterized this phenomenon by using extracellular recordings in brain slices to examine whether DIR is linked to phosphatidylinositol (PI) or adenylate cyclase (AC) second-messenger pathways. A D1-like dopaminergic agonist associated with PI turnover (SKF83959), but not one linked to AC (SKF83822), promoted reversal of inhibition produced by quinpirole, a dopamine D2-selective agonist. Other neurotransmitter receptors linked to PI turnover include serotonin 5-HT(2), α(1)-adrenergic, neurotensin, and group I metabotropic glutamate (mGlu) receptors. Both serotonin and neurotensin produced significant reversal of quinpirole inhibition, but agonists of α(1)-adrenergic and group I mGlu receptors failed to significantly reverse quinpirole inhibition. These results indicate that some agonists that stimulate PI turnover can facilitate desensitization of D2 receptors but that there may be other factors in addition to PI that control that interaction.

Ethanol blocks the reversal of prolonged dopamine inhibition of dopaminergic neurons of the ventral tegmental area.

BACKGROUND: Dopaminergic (DAergic) neurons of the ventral tegmental area (VTA) are important for the rewarding and reinforcing properties of alcohol and other drugs of abuse. Regulation of the firing of DAergic VTA neurons is controlled by a number of factors, including autoregulation of firing by D2 dopamine (DA) receptors. The inhibitory effects of DA on these neurons exhibit concentration- and time-dependent desensitization, which we have termed dopamine inhibition reversal (DIR), as it requires concurrent stimulation of D1/D5 and D2 receptors. METHODS: Extracellular recording of DAergic VTA neurons in brain slices was used to test the effects of ethanol (EtOH) (10 to 80 mM) on DIR. RESULTS: DIR was reduced by concentrations of EtOH as low as 10 mM and was blocked by higher EtOH concentrations. In addition, as we have shown that reversal of inhibition by the selective D2 agonist quinpirole can be observed in the presence of an activator of protein kinase C (PKC), we tested whether EtOH could antagonize the reversal of quinpirole inhibition in the presence of phorbol 12-myristate 13-acetate (PMA). EtOH (80 mM) blocked the reversal of quinpirole seen in the presence of PMA, suggesting that the antagonism of DIR by EtOH is owing to an action at a stage in the mechanism at or distal to PKC. Once achieved, DIR is not antagonized by EtOH. CONCLUSIONS: The blockade by relatively low concentrations of EtOH of DIR may play an important role in the spectrum of action of EtOH on DAergic neurons of the VTA and may be important in the acute and chronic actions of EtOH on the excitability of these brain reward/reinforcement neurons.

Differential Effects of Toluene and Ethanol on Dopaminergic Neurons of the Ventral Tegmental Area.

Drugs of abuse increase the activity of dopaminergic neurons of the ventral tegmental area (VTA), and output from the VTA is critical for both natural and drug-induced reward and reinforcement. Ethanol and the abused inhalant toluene both enhance VTA neuronal firing, but the mechanisms of this effect is not fully known. In this study, we used extracellular recordings to compare the actions of toluene and ethanol on DA VTA neurons. Both ethanol and toluene increased the firing rate of DA neurons, although toluene was ~100 times more potent than ethanol. The mixed ion channel blocker quinine (100 µM) blocked the increases in firing produced by ethanol and toluene, indicating some similarity in mechanisms of excitation. A mixture of antagonists of GABA and cholinergic receptors did not prevent toluene-induced or ethanol-induced excitation, and toluene-induced excitation was not altered by co-administration of ethanol, suggesting independent mechanisms of excitation for ethanol and toluene. Concurrent blockade of NMDA, AMPA, and metabotropic glutamate receptors enhanced the excitatory effect of toluene while having no significant effect on ethanol excitation. Nicotine increased firing of DA VTA neurons, and this was blocked by the nicotinic antagonist mecamylamine (1 µM). Mecamylamine did not alter ethanol or toluene excitation of firing but the muscarinic antagonist atropine (5 µM) or a combination of GABA antagonists (bicuculline and CGP35348, 10 µM each) reduced toluene-induced excitation without affecting ethanol excitation. The Ih current blocker ZD7288 abolished the excitatory effect of toluene but unlike the block of ethanol excitation, the effect of ZD7288 was not reversed by the GIRK channel blocker barium, but was reversed by GABA antagonists. These results demonstrate that the excitatory effects of ethanol and toluene have some similarity, such as block by quinine and ZD7288, but also indicate that there are important differences between these two drugs in their modulation by glutamatergic, cholinergic, and GABAergic receptors. These findings provide important information regarding the actions of abused inhalants on central reward pathways, and suggest that regulation of the activation of central dopamine pathways by ethanol and toluene partially overlap.

Dopamine D2 receptor desensitization by dopamine or corticotropin releasing factor in ventral tegmental area neurons is associated with increased glutamate release.

Neurons of the ventral tegmental area (VTA) are the source of dopaminergic (DAergic) input to important brain regions related to addiction. Prolonged exposure of these VTA neurons to moderate concentrations of dopamine (DA) causes a time-dependent decrease in DA-induced inhibition, a complex desensitization called DA inhibition reversal (DIR). DIR is mediated by conventional protein kinase C (cPKC) through concurrent stimulation of D2 and D1-like DA receptors, or by D2 stimulation concurrent with activation of some Gq-linked receptors. Corticotropin releasing factor (CRF) acts via Gq, and can modulate glutamater neurotransmission in the VTA. In the present study, we used brain slice electrophysiology to characterize the interaction of DA, glutamate antagonists, and CRF agonists in the induction and maintenance of DIR in the VTA. Glutamate receptor antagonists blocked induction but not maintenance of DIR. Putative blockers of neurotransmitter release and store-operated calcium channels blocked and reversed DIR. CRF and the CRF agonist urocortin reversed inhibition produced by the D2 agonist quinpirole, consistent with our earlier work indicating that Gq activation reverses quinpirole-mediated inhibition. In whole cell recordings, the combination of urocortin and quinpirole, but not either agent alone, increased spontaneous excitatory postsynaptic currents (sEPSCs) in VTA neurons. Likewise, the combination of a D1-like receptor agonist and quinpirole, but not either agent alone, increased sEPSCs in VTA neurons. In summary, desensitization of D2 receptors induced by dopamine or CRF on DAergic VTA neurons is associated with increased glutamatergic signaling in the VTA.

Suppression of Gq Function Using Intra-Pipette Delivery of shRNA during Extracellular Recording in the Ventral Tegmental Area.

Selective suppression of protein function in the brain can be achieved using specific silencing RNAs administered in vivo. A viral delivery system is often employed to transfect neurons with small hairpin RNA (shRNA) directed against specific proteins, and intervals of several days are allowed between microinjection of the shRNA-containing virus into the brain and experiments to assess suppression of gene function. Here we report studies using extracellular recording of dopaminergic neurons of the ventral tegmental area (DA VTA neurons) recorded in brain slices in which lentivirus containing shRNA directed against Gq was included in the recording pipette, and suppression of Gq-related function was observed within the time frame of the recording. The action of neurotensin (NT) is associated with activation of Gq, and the firing rate of DA VTA neurons is increased by NT. With shRNA directed against Gq in the pipette, there was a significant reduction of NT excitation within 2 h. Likewise, time-dependent dopamine desensitization, which we have hypothesized to be Gq-dependent, was not observed when shRNA directed against Gq was present in the pipette and dopamine was tested 2 h after initiation of recording. As the time interval (2 h) is relatively short, we tested whether blockade of protein synthesis with cycloheximide delivered via the recording pipette would alter Gq-linked responses similarly. Both NT-induced excitation and dopamine desensitization were inhibited in the presence of cycloheximide. Inclusion of shRNA in the recording pipette may be an efficient and selective way to dampen responses linked to Gq, and, more generally, the use of lentiviral-packaged shRNA in the recording pipette is a means to produce selective inhibition of the function of specific proteins in experiments.

Phorbol ester reduces ethanol excitation of dopaminergic neurons of the ventral tegmental area: involvement of protein kinase C theta.

Neurons of the ventral tegmental area (VTA) play a key role in the rewarding and reinforcing effects of drugs of abuse, including alcohol. Ethanol directly increases the firing rate of dopaminergic (DAergic) VTA neurons, but modulation of the firing rate of DAergic VTA neurons can be controlled by a number of factors, including some that are under the control of protein kinase C (PKC). Application of phorbol esters activates PKC and the present study assessed the effect of a phorbol ester, phorbol 12-myristate 13-acetate (PMA), on ethanol-induced excitation of DA VTA neurons. Ethanol-induced excitation of DAergic VTA neurons was reduced significantly in the presence of PMA. This action of PMA was antagonized by chelerythrine chloride, a non-selective antagonist of PKC, but not by moderate concentrations of antagonists of conventional PKC isoforms (Gö6976 and Gö6983). A PKC δ/θ inhibitor antagonized PMA-induced reduction of ethanol excitation. Since PKCδ antagonist Gö6983 did not antagonize the effect of PMA on ethanol excitation, the PMA reduction of ethanol excitation is most likely to be mediated by PKCθ. Antagonists of intracellular calcium pathways were ineffective in antagonizing PMA action on ethanol excitation, consistent with the lack of calcium dependence of PKCθ. In summary, ethanol-induced excitation of VTA neurons is attenuated in the presence of PMA, and this attenuation appears to be mediated by PKCθ. This novel mechanism for interfering with ethanol activation of reward-related neurons could provide a new target for pharmacotherapy to ameliorate alcoholism.

Hyposensitivity to gamma-aminobutyric acid in the ventral tegmental area during alcohol withdrawal: reversal by histone deacetylase inhibitors.

Putative dopaminergic (pDAergic) ventral tegmental area (VTA) neurons have an important role in alcohol addiction. Acute ethanol increases the activity of pDAergic neurons, and withdrawal from repeated ethanol administration produces a decreased sensitivity of pDAergic VTA neurons to GABA. Recent studies show that behavioral changes induced by chronic alcohol are reversed by inhibitors of histone deacetylases (HDACs). Whether HDAC-induced histone modifications regulate changes in GABA sensitivity of VTA pDAergic neurons during withdrawal is unknown. Here, we investigated modulation of withdrawal-induced changes in GABA sensitivity of pDAergic VTA neurons by HDAC inhibitors (HDACi), and also measured the levels of HDAC2, histone (H3-K9) acetylation, and GABA-Aα1 receptor (GABA (A-α1) R) subunit in VTA during ethanol withdrawal. Mice were injected intraperitoneally (ip) with either ethanol (3.5 g/kg) or saline twice daily for 3 weeks. In recordings from pDAergic VTA neurons in brain slices from ethanol-withdrawn mice, sensitivity to GABA (50-500 µM) was reduced. In brain slices from ethanol-withdrawn mice incubated with the HDACi SAHA (vorinostat) or trichostatin A (TSA) for 2 h, the hyposensitivity of pDAergic VTA neurons to GABA was significantly attenuated. There was no effect of TSA or SAHA on GABA sensitivity of pDAergic VTA neurons from saline-treated mice. In addition, ethanol withdrawal was associated with an increase in levels of HDAC2 and a decrease in histone (H3-K9) acetylation and levels of GABA (A-α1) R subunits in the VTA. Therefore, blockade of upregulation of HDAC2 by HDACi normalizes GABA hyposensitivity of pDAergic neurons developed during withdrawal after chronic ethanol treatment, which suggests the possibility that inhibition of HDACs can reverse ethanol-induced neuroadaptational changes in reward circuitry.

Reversal of dopamine inhibition of dopaminergic neurons of the ventral tegmental area is mediated by protein kinase C.

Adaptation of putative dopaminergic (pDA) neurons in the ventral tegmental area (VTA) to drugs of abuse may alter information processing related to reward and reinforcement and is an important factor in the development of addiction. We have demonstrated that prolonged increases in the concentration of dopamine (DA) result in a time-dependent decrease in sensitivity of pDA neurons to DA, which we termed DA inhibition reversal (DIR). In this study, we used extracellular recordings to examine factors mediating DIR. A 40 min administration of DA (2.5-10 µM), but not the DA D2 receptor agonist quinpirole (50-200 nM), resulted in inhibition of neuronal firing followed by DIR. In the presence of 100 nM cocaine, inhibition followed by DIR was seen with much lower DA concentrations. Reversal of quinpirole inhibition could be induced by an activator of protein kinase C, but not of protein kinase A. Inhibitors of protein kinase C or phospholipase C blocked the development of DIR. Disruption of intracellular calcium release also prevented DIR. Reduction of extracellular calcium or inhibition of store-operated calcium entry blocked DIR, but the L-type calcium channel blocker nifedipine did not. DIR was age-dependent and not seen in pDA VTA neurons from rat pups younger than 15 days postnatally. Our data indicate that DIR is mediated by protein kinase C, and implicate a conventional protein kinase C. This characterization of DIR gives insight into the regulation of autoinhibition of pDA VTA neurons, and the resulting long-term alteration in information processing related to reward and reinforcement.

Treatment with a novel hemigramicidin-TEMPO conjugate prolongs survival in a rat model of lethal hemorrhagic shock.

OBJECTIVE: We sought to develop a therapeutic agent that would permit prolongation of survival in rats subjected to lethal hemorrhagic shock (HS), even in the absence of resuscitation with asanguinous fluids or blood. METHODS AND RESULTS: We synthesized a series of compounds that consist of the electron scavenger and superoxide dismutase mimic, 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-NH2-TEMPO), conjugated to fragments and analogs of the membrane-active cyclopeptide antibiotic, gramicidin S. Using an in vivo assay, wherein isolated intestinal segments were loaded inside the lumen with various test compounds, we studied these compounds for their ability to prevent ileal mucosal barrier dysfunction induced by subjecting rats to profound HS for 2 hours. The most active compound in this assay, XJB-5-131, ameliorated peroxidation of the mitochondrial phospholipid, cardiolipin, in ileal mucosal samples from rats subjected to HS. XJB-5-131 also ameliorated HS-induced activation of the pro-apoptotic enzymes, caspases 3 and 7, in ileal mucosa. Intravenous treatment with XJB-5-131 (2 micromol/kg) significantly prolonged the survival of rats subjected to profound blood loss (33.5 mL/kg) despite administration of only a minimal volume of crystalloid solution (2.8 mL/kg) and the absence of blood transfusion. CONCLUSION: These data support the view that mitochondrially targeted electron acceptors and SOD mimics are potentially valuable therapeutics for the treatment of serious acute conditions, such as HS, which are associated with marked tissue ischemia.