Dopamine Receptors Differentially Control Binge Alcohol Drinking-Mediated Synaptic Plasticity of the Core Nucleus Accumbens Direct and Indirect Pathways.

Binge alcohol drinking, a behavior characterized by rapid repeated alcohol intake, is most prevalent in young adults and is a risk factor for excessive alcohol consumption and alcohol dependence. Although the alteration of synaptic plasticity is thought to contribute to this behavior, there is currently little evidence that this is the case. We used drinking in the dark (DID) as a model of binge alcohol drinking to assess its effects on spike timing-dependent plasticity (STDP) in medium spiny neurons (MSNs) of the core nucleus accumbens (NAc) by combining patch-clamp recordings with calcium imaging and optogenetics. After 2 weeks of daily alcohol binges, synaptic plasticity was profoundly altered. STDP in MSNs expressing dopamine D1 receptors shifted from spike-timing-dependent long-term depression (tLTD), the predominant form of plasticity in naive male mice, to spike-timing-dependent long-term potentiation (tLTP) in DID mice, an effect that was totally reversed in the presence of 4 µm SCH23390, a dopamine D1 receptor antagonist. In MSNs presumably expressing dopamine D2 receptors, tLTP, the main form of plasticity in naive mice, was inhibited in DID mice. Interestingly, 1 µm sulpiride, a D2 receptor antagonist, restored tLTP. Although we observed no alterations of AMPA and NMDA receptor properties, we found that the AMPA/NMDA ratio increased at cortical and amygdaloid inputs but not at hippocampal inputs. Also, DID effects on STDP were accompanied by lower dendritic calcium transients. These data suggest that the role of dopamine in mediating the effects of binge alcohol drinking on synaptic plasticity of NAc MSNs differs markedly whether these neurons belong to the direct or indirect pathways.SIGNIFICANCE STATEMENT We examined the relationship between binge alcohol drinking and spike timing-dependent plasticity in nucleus accumbens (NAc) neurons. We found that repeated drinking bouts modulate differently synaptic plasticity in medium spiny neurons of the accumbens direct and indirect pathways. While timing-dependent long-term depression switches to long-term potentiation (LTP) in the former, timing-dependent LTP is inhibited in the latter. These effects are not accompanied by changes in AMPA and NMDA receptor properties at cortical, amygdaloid, and hippocampal synapses. Interestingly, dopamine D1 and D2 receptor antagonists have opposite effects on plasticity. Our data show that whether core NAc medium spiny neurons belong to the direct or indirect pathways determines the form of spike timing-dependent plasticity (STDP), the manner by which STDP responds to binge alcohol drinking, and its sensitivity to dopamine receptor antagonists.

The Sodium Channel ß4 Auxiliary Subunit Selectively Controls Long-Term Depression in Core Nucleus Accumbens Medium Spiny Neurons.

Voltage-gated sodium channels are essential for generating the initial rapid depolarization of neuronal membrane potential during action potentials (APs) that enable cell-to-cell communication, the propagation of signals throughout the brain, and the induction of synaptic plasticity. Although all brain neurons express one or several variants coding for the core pore-forming sodium channel α subunit, the expression of the ß (ß1-4) auxiliary subunits varies greatly. Of particular interest is the ß4 subunit, encoded by the Scn4b gene, that is highly expressed in dorsal and ventral (i.e., nucleus accumbens - NAc) striata compared to other brain regions, and that endows sodium channels with unique gating properties. However, its role on neuronal activity, synaptic plasticity, and behaviors related to drugs of abuse remains poorly understood. Combining whole-cell patch-clamp recordings with two-photon calcium imaging in Scn4b knockout (KO) and knockdown mice, we found that Scn4b altered the properties of APs in core accumbens medium spiny neurons (MSNs). These alterations are associated with a reduction of the probability of MSNs to evoke spike-timing-dependent long-term depression (tLTD) and a reduced ability of backpropagating APs to evoke dendritic calcium transients. In contrast, long-term potentiation (tLTP) remained unaffected. Interestingly, we also showed that amphetamine-induced locomotor activity was significantly reduced in male Scn4b KO mice compared to wild-type controls. Taken together, these data indicate that the Scn4b subunit selectively controls tLTD by modulating dendritic calcium transients evoked by backpropagating APs.

Modulation of ethanol reward sensitivity by nicotinic acetylcholine receptors containing the α6 subunit.

The prevalent co-abuse of nicotine and alcohol suggests a common neural mechanism underlying the actions of the two drugs. Nicotine, the addictive component of tobacco, activates nicotinic acetylcholine receptors (nAChRs) containing the α6 subunit (α6* nAChRs) in dopaminergic (DAergic) neurons of the ventral tegmental area (VTA), a region known to be crucial for drug reward. Recent evidence suggests that ethanol may potentiate ACh activation of these receptors as well, although whether α6* nAChR expression is necessary for behavioral effects of acute ethanol exposure is unknown. We compared binge-like ethanol consumption and ethanol reward sensitivity between knockout (KO) mice that do not express chrna6 (the gene encoding the α6 nAChR subunit, the α6 KO line) and wild-type (WT) littermates using the Drinking-in-the-Dark (DID) and Conditioned Place Preference (CPP) assay, respectively. In the DID assay, α6 KO female and male mice consumed ethanol similarly to WT mice at all concentrations tested. In the CPP assay, 2.0-g/kg and 3.0-g/kg, but not 0.5-mg/kg, ethanol conditioned a place preference in WT female and male mice, whereas only 2.0-g/kg ethanol conditioned a place preference in α6 KO mice. Acute challenge with ethanol reduced locomotor activity, an effect that developed tolerance with repeated injections, similarly between genotypes in both female and male mice. Together, these data indicate that expression of α6* nAChRs is not required for binge-like ethanol consumption and reward, but modulate sensitivity to the rewarding properties of the drug.

FT-IR spectroscopy and density functional theory calculations of 13C isotopologues of the helical peptide Z-Aib6-OtBu.

Isotope-edited FT-IR spectroscopy is a combined synthetic and spectroscopic method used to characterize local (e.g., residue-level) vibrational environments of biomolecules. We have prepared the 3(10) helical peptide Z-Aib6-OtBu and seven (13)C-enriched analogues that vary only in the number and position(s) of (13)C═O isotopic enrichment. FT-IR spectra of these eight peptides solvated in the nonpolar aprotic solvent dichloromethane have been collected and compared to frequency, intensity, and normal mode results of DFT calculations. Single (13)C enrichment of amide functional groups tends to localize amide I vibrational eigenmodes, providing residue-specific information regarding the local environment (e.g., hydrogen bonding or solvent exposure) of the peptide bond. Double (13)C enrichment of Z-Aib6-OtBu allows for examination of interamide coupling between two labeled amide functional groups, providing experimental evidence of interamide coupling in the context of 3(10) helical structure. Although the calculated and observed interamide couplings of Z-Aib6-OtBu are a few cm(-1) and less, the eight peptides exhibit distinct infrared spectra, revealing details of interamide coupling and residue level vibrational environments.

Neuronal nicotinic acetylcholine receptors: common molecular substrates of nicotine and alcohol dependence.

Alcohol and nicotine are often co-abused. As many as 80-95% of alcoholics are also smokers, suggesting that ethanol and nicotine, the primary addictive component of tobacco smoke, may functionally interact in the central nervous system and/or share a common mechanism of action. While nicotine initiates dependence by binding to and activating neuronal nicotinic acetylcholine receptors (nAChRs), ligand-gated cation channels normally activated by endogenous acetylcholine (ACh), ethanol is much less specific with the ability to modulate multiple gene products including those encoding voltage-gated ion channels, and excitatory/inhibitory neurotransmitter receptors. However, emerging data indicate that ethanol interacts with nAChRs, both directly and indirectly, in the mesocorticolimbic dopaminergic (DAergic) reward circuitry to affect brain reward systems. Like nicotine, ethanol activates DAergic neurons of the ventral tegmental area (VTA) which project to the nucleus accumbens (NAc). Blockade of VTA nAChRs reduces ethanol-mediated activation of DAergic neurons, NAc DA release, consumption, and operant responding for ethanol in rodents. Thus, ethanol may increase ACh release into the VTA driving activation of DAergic neurons through nAChRs. In addition, ethanol potentiates distinct nAChR subtype responses to ACh and nicotine in vitro and in DAergic neurons. The smoking cessation therapeutic and nAChR partial agonist, varenicline, reduces alcohol consumption in heavy drinking smokers and rodent models of alcohol consumption. Finally, single nucleotide polymorphisms in nAChR subunit genes are associated with alcohol dependence phenotypes and smoking behaviors in human populations. Together, results from pre-clinical, clinical, and genetic studies indicate that nAChRs may have an inherent role in the abusive properties of ethanol, as well as in nicotine and alcohol co-dependence.

Nicotinic acetylcholine receptors containing the α4 subunit modulate alcohol reward.

BACKGROUND: Nicotine and alcohol are the two most co-abused drugs in the world, suggesting a common mechanism of action might underlie their rewarding properties. Although nicotine elicits reward by activating ventral tegmental area dopaminergic (DAergic) neurons via high-affinity neuronal nicotinic acetylcholine receptors (nAChRs), the mechanism by which alcohol activates these neurons is unclear. METHODS: Because most high-affinity nAChRs expressed in ventral tegmental area DAergic neurons contain the α4 subunit, we measured ethanol-induced activation of DAergic neurons in midbrain slices from two complementary mouse models, an α4 knock-out (KO) mouse line and a knock-in line (Leu9'Ala) expressing α4 subunit-containing nAChRs hypersensitive to agonist compared with wild-type (WT). Activation of DAergic neurons by ethanol was analyzed with both biophysical and immunohistochemical approaches in midbrain slices. The ability of alcohol to condition a place preference in each mouse model was also measured. RESULTS: At intoxicating concentrations, ethanol activation of DAergic neurons was significantly reduced in α4 KO mice compared with WT. Conversely, in Leu9'Ala mice, DAergic neurons were activated by low ethanol concentrations that did not increase activity of WT neurons. In addition, alcohol potentiated the response to ACh in DAergic neurons, an effect reduced in α4 KO mice. Rewarding alcohol doses failed to condition a place preference in α4 KO mice, paralleling alcohol effects on DAergic neuron activity, whereas a sub-rewarding alcohol dose was sufficient to condition a place preference in Leu9'Ala mice. CONCLUSIONS: Together, these data indicate that nAChRs containing the α4 subunit modulate alcohol reward.

Viral cell death inhibitor MC159 enhances innate immunity against vaccinia virus infection.

Viral inhibitors of host programmed cell death (PCD) are widely believed to promote viral replication by preventing or delaying host cell death. Viral FLIPs (Fas-linked ICE-like protease [FLICE; caspase-8]-like inhibitor proteins) are potent inhibitors of death receptor-induced apoptosis and programmed necrosis. Surprisingly, transgenic expression of the viral FLIP MC159 from molluscum contagiosum virus (MCV) in mice enhanced rather than inhibited the innate immune control of vaccinia virus (VV) replication. This effect of MC159 was specifically manifested in peripheral tissues such as the visceral fat pad, but not in the spleen. VV-infected MC159 transgenic mice mounted an enhanced innate inflammatory reaction characterized by increased expression of the chemokine CCL-2/MCP-1 and infiltration of γδ T cells into peripheral tissues. Radiation chimeras revealed that MC159 expression in the parenchyma, but not in the hematopoietic compartment, is responsible for the enhanced innate inflammatory responses. The increased inflammation in peripheral tissues was not due to resistance of lymphocytes to cell death. Rather, we found that MC159 facilitated Toll-like receptor 4 (TLR4)- and tumor necrosis factor (TNF)-induced NF-κB activation. The increased NF-κB responses were mediated in part through increased binding of RIP1 to TNFRSF1A-associated via death domain (TRADD), two crucial signal adaptors for NF-κB activation. These results show that MC159 is a dual-function immune modulator that regulates host cell death as well as NF-κB responses by innate immune signaling receptors.

Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation.

Programmed necrosis is a form of caspase-independent cell death whose molecular regulation is poorly understood. The kinase RIP1 is crucial for programmed necrosis, but also mediates activation of the prosurvival transcription factor NF-kappaB. We postulated that additional molecules are required to specifically activate programmed necrosis. Using a RNA interference screen, we identified the kinase RIP3 as a crucial activator for programmed necrosis induced by TNF and during virus infection. RIP3 regulates necrosis-specific RIP1 phosphorylation. The phosphorylation of RIP1 and RIP3 stabilizes their association within the pronecrotic complex, activates the pronecrotic kinase activity, and triggers downstream reactive oxygen species production. The pronecrotic RIP1-RIP3 complex is induced during vaccinia virus infection. Consequently, RIP3(-/-) mice exhibited severely impaired virus-induced tissue necrosis, inflammation, and control of viral replication. Our findings suggest that RIP3 controls programmed necrosis by initiating the pronecrotic kinase cascade, and that this is necessary for the inflammatory response against virus infections.