Conscious intention and human action: Review of the rise and fall of the readiness potential and Libet's clock.

Is consciousness-the subjective awareness of the sensations, perceptions, beliefs, desires, and intentions of mental life-a genuine cause of human action or a mere impotent epiphenomenon accompanying the brain's physical activity but utterly incapable of making anything actually happen? This article will review the history and current status of experiments and commentary related to Libet's influential paper (Brain 106:623-664, 1983) whose conclusion "that cerebral initiation even of a spontaneous voluntary act …can and usually does begin unconsciously" has had a huge effect on debate about the efficacy of conscious intentions. Early (up to 2008) and more recent (2008 on) experiments replicating and criticizing Libet's conclusions and especially his methods will be discussed, focusing especially on recent observations that the readiness potential (RP) may only be an "artifact of averaging" and that, when intention is measured using "tone probes," the onset of intention is found much earlier and often before the onset of the RP. Based on these findings, Libet's methodology was flawed and his results are no longer valid reasons for rejecting Fodor's "good old commonsense belief/desire psychology" that "my wanting is causally responsible for my reaching.".

PARP inhibition in vivo blocks alcohol-induced brain neurodegeneration and neuroinflammatory cytosolic phospholipase A2 elevations.

Chronic alcoholism promotes brain damage that impairs memory and cognition. High binge alcohol levels in adult rats also cause substantial neurodamage to memory-linked regions, notably, the hippocampus (HC) and entorhinal cortex (ECX). Concurrent with neurodegeneration, alcohol elevates poly (ADP-ribose) polymerase-1 (PARP-1) and cytosolic phospholipase A2 (cPLA2) levels. PARP-1 triggers necrosis when excessively activated, while cPLA2 liberates neuroinflammatory ω-6 arachidonic acid. Inhibitors of PARP exert in vitro neuroprotection while suppressing cPLA2 elevations in alcohol-treated HC-ECX slice cultures. Here, we examined in vivo neuroprotection and cPLA2 suppression by the PARP inhibitor, veliparib, in a recognized adult rat model of alcohol-binging. Adult male rats received Vanilla Ensure containing alcohol (ethanol, 7.1 ±â€¯0.3 g/kg/day), or control (dextrose) ±â€¯veliparib (25 mg/kg/day), by gavage 3x daily for 4 days. Rats were sacrificed on the morning after the final binge. HC and ECX neurodegeneration was assessed in fixed sections by Fluoro-Jade B (FJB) staining. Dorsal HC, ventral HC, and ECX cPLA2 levels were quantified by immunoblotting. Like other studies using this model, alcohol binges elevated FJB staining in the HC (dentate gyrus) and ECX, indicating neurodegeneration. Veliparib co-treatment significantly reduced dentate gyrus and ECX neurodegeneration by 79% and 66%, respectively. Alcohol binges increased cPLA2 in the ventral HC by 34% and ECX by 72%, which veliparib co-treatment largely prevented. Dorsal HC cPLA2 levels remained unaffected by alcohol binges, consistent with negligible FJB staining in this brain region. These in vivo results support an emerging key role for PARP in binge alcohol-induced neurodegeneration and cPLA2-related neuroinflammation.

Ethanol preconditioning of rat cerebellar cultures targets NMDA receptors to the synapse and enhances peroxiredoxin 2 expression.

Epidemiological studies indicate that light-moderate alcohol (ethanol) consumers tend to have reduced risks of cognitive impairment and progression to dementia during aging. Exploring possible mechanisms, we previously found that moderate ethanol preconditioning (MEP, 20-30mM) of rat brain cultures for several days instigated neuroprotection against ß-amyloid peptides. Our biochemical evidence implicated the NMDA receptor (NMDAR) as a potential neuroprotective "sensor", specifically via synaptic NMDAR signaling. It remains unclear how ethanol modulates the receptor and its downstream targets to engender neuroprotection. Here we confirm with deconvolution microscopy that MEP of rat mixed cerebellar cultures robustly increases synaptic NMDAR localization. Phospho-activation of the non-receptor tyrosine kinases Src and Pyk2, known to be linked to synaptic NMDAR, is also demonstrated. Additionally, the preconditioning enhances levels of an antioxidant protein, peroxiredoxin 2 (Prx2), reported to be downstream of synaptic NMDAR signaling, and NMDAR antagonism with memantine (earlier found to abrogate MEP neuroprotection) blocks the Prx2 elevations. To further link Prx2 with antioxidant-based neuroprotection, we circumvented the ethanol preconditioning-NMDAR pathway by pharmacologically increasing Prx2 with the naturally-occurring cruciferous compound, 3H-1,2-dithiole-3-thione (D3T). Thus, D3T pretreatment elevated Prx2 expression to a similar extent as MEP, while concomitantly preventing ß-amyloid neurotoxicity; D3T also protected the cultures from hydrogen peroxide toxicity. The findings support a mechanism that couples synaptic NMDAR signaling, Prx2 expression and augmented antioxidant defenses in ethanol preconditioning-induced neuroprotection. That this mechanism can be emulated by a cruciferous vegetable constituent suggests that such naturally-occurring "neutraceuticals" may be useful in therapy for oxidative stress-related dementias.

Neuroinflammation and neurodegeneration in adult rat brain from binge ethanol exposure: abrogation by docosahexaenoic acid.

Evidence that brain edema and aquaporin-4 (AQP4) water channels have roles in experimental binge ethanol-induced neurodegeneration has stimulated interest in swelling/edema-linked neuroinflammatory pathways leading to oxidative stress. We report here that neurotoxic binge ethanol exposure produces comparable significant effects in vivo and in vitro on adult rat brain levels of AQP4 as well as neuroinflammation-linked enzymes: key phospholipase A2 (PLA2) family members and poly (ADP-ribose) polymerase-1 (PARP-1). In adult male rats, repetitive ethanol intoxication (3 gavages/d for 4 d, ∼ 9 g/kg/d, achieving blood ethanol levels ∼ 375 mg/dl; "Majchrowicz" model) significantly increased AQP4, Ca+2-dependent PLA2 GIVA (cPLA2), phospho-cPLA2 GIVA (p-cPLA2), secretory PLA2 GIIA (sPLA2) and PARP-1 in regions incurring extensive neurodegeneration in this model--hippocampus, entorhinal cortex, and olfactory bulb--but not in two regions typically lacking neurodamage, frontal cortex and cerebellum. Also, ethanol reduced hippocampal Ca+2-independent PLA2 GVIA (iPLA2) levels and increased brain "oxidative stress footprints" (4-hydroxynonenal-adducted proteins). For in vitro studies, organotypic cultures of rat hippocampal-entorhinocortical slices of adult age (∼ 60 d) were ethanol-binged (100 mM or ∼ 450 mg/dl) for 4 d, which augments AQP4 and causes neurodegeneration (Collins et al. 2013). Reproducing the in vivo results, cPLA2, p-cPLA2, sPLA2 and PARP-1 were significantly elevated while iPLA2 was decreased. Furthermore, supplementation with docosahexaenoic acid (DHA; 22:6n-3), known to quell AQP4 and neurodegeneration in ethanol-treated slices, blocked PARP-1 and PLA2 changes while counteracting endogenous DHA reduction and increases in oxidative stress footprints (3-nitrotyrosinated proteins). Notably, the PARP-1 inhibitor PJ-34 suppressed binge ethanol-dependent neurodegeneration, indicating PARP upstream involvement. The results with corresponding models support involvement of AQP4- and PLA2-associated neuroinflammatory pro-oxidative pathways in the neurodamage, with potential regulation by PARP-1 as well. Furthermore, DHA emerges as an effective inhibitor of these binge ethanol-dependent neuroinflammatory pathways as well as associated neurodegeneration in adult-age brain.

Alcohol, phospholipase A2-associated neuroinflammation, and ω3 docosahexaenoic acid protection.

Chronic alcohol (ethanol) abuse causes neuroinflammation and brain damage that can give rise to alcoholic dementia. Insightfully, Dr. Albert Sun was an early proponent of oxidative stress as a key factor in alcoholism-related brain deterioration. In fact, oxidative stress has proven to be critical to the hippocampal and temporal cortical neurodamage resulting from repetitive "binge" alcohol exposure in adult rat models. Although the underlying mechanisms are uncertain, our immunoelectrophoretic and related assays in binge alcohol experiments in vivo (adult male rats) and in vitro (rat organotypic hippocampal-entorhinal cortical slice cultures) have implicated phospholipase A(2) (PLA(2))-activated neuroinflammatory pathways, release of pro-oxidative arachidonic acid (20:4 ω6), and elevated oxidative stress adducts (i.e., 4-hydroxynonenal-protein adducts). Also, significantly increased by the binge alcohol treatments was aquaporin-4 (AQP4), a water channel enriched in astrocytes that, when augmented, may trigger brain (esp. cellular) edema and neuroinflammation; of relevance, glial swelling is known to provoke increased PLA(2) activities or levels. Concomitant with PLA(2) activation, the results have further implicated binge alcohol-elevated poly (ADP-ribose) polymerase-1 (PARP-1), an oxidative stress-responsive DNA repair enzyme linked to parthanatos, a necrotic-like neuronal death process. Importantly, supplementation of the brain slice cultures with docosahexaenoic acid (22:6 ω3) exerted potent suppression of the induced changes in PLA(2) isoforms, AQP4, PARP-1 and oxidative stress footprints, and prevention of the binge alcohol neurotoxicity, by as yet unknown mechanisms. These neuroinflammatory findings from our binge alcohol studies and supportive rat binge studies in the literature are reviewed.

Phospholipase A2, oxidative stress, and neurodegeneration in binge ethanol-treated organotypic slice cultures of developing rat brain.

BACKGROUND: Brain neurodamage from chronic binge ethanol (EtOH) exposure is linked to neuroinflammation and associated oxidative stress. Using rat organotypic hippocampal-entorhinal cortical (HEC) slice cultures of developing brain age, we reported that binge EtOH promotes release of a neuroinflammatory instigator, arachidonic acid (AA), concomitant with neurodegeneration, and that mepacrine, a global inhibitor of phospholipase A2 (PLA2) enzymes mobilizing AA from phospholipids, is neuroprotective. Here, we sought with binge EtOH-treated HEC cultures to establish that PLA2 activity is responsible in part for significant oxidative stress and to ascertain the PLA2 families responsible for AA release and neurodegeneration. METHODS: HEC slices, prepared from 1-week-old rats and cultured 2 to 2.5 weeks, were exposed to 100 mM EtOH over 6 successive days, with 4 daytime "withdrawals" (no EtOH). Brain 3-nitrotyrosinated (3-NT)- and 4-hydroxy-2-nonenal (4-HNE)-adducted proteins, oxidative stress footprints, were immunoassayed on days 3 through 6, and mepacrine's effect was determined on day 6. The effects of specific PLA2 inhibitors on neurodegeneration (propidium iodide staining) and AA release (ELISA levels in media) in the cultures were then determined. Also, the effect of JZL184, an inhibitor of monoacylglycerol lipase (MAGL) which is reported to mobilize AA from endocannabinoids during neuroinflammatory insults, was examined. RESULTS: 3-NT- and 4-HNE-adducted proteins were significantly increased by the binge EtOH exposure, consistent with oxidative stress, and mepacrine prevented the increases. The PLA2 inhibitor results implicated secretory PLA2 (group II sPLA2) and to some extent Ca(2+) -independent cytosolic PLA2 (group VI iPLA2) in binge EtOH-induced neurotoxicity and in AA release, but surprisingly, Ca(2+) -dependent cytosolic PLA2 (group IV cPLA2) did not appear important. Furthermore, unlike PLA2 inhibition, MAGL inhibition failed to prevent the neurodegeneration. CONCLUSIONS: In these developing HEC slice cultures, pro-oxidative signaling via sPLA2 and iPLA2, but not necessarily cPLA2 or MAGL, is involved in EtOH neurotoxicity. This study provides further insights into neuroinflammatory phospholipase signaling and oxidative stress underlying binge EtOH-induced neurodegeneration in developing (adolescent age) brain in vitro.

Docosahexaenoic acid (DHA) prevents binge ethanol-dependent aquaporin-4 elevations while inhibiting neurodegeneration: experiments in rat adult-age entorhino-hippocampal slice cultures.

Repetitive binge intoxication with ethanol (alcohol) in adult rats, mimicking chronic ethanol abuse in alcoholics, causes trauma-like brain edema and relatively selective neurodegeneration of hippocampal dentate granule cells and pyramidal neurons in the temporal cortex (especially entorhinal cortex). We have now modeled the aspects of this type of acquired brain damage in vitro with rat entorhino-hippocampal slice cultures of adult brain age (62 ± 3 days). When sequentially treated (four 16-h overnight exposures) with 100 mM ethanol, the slices display elevated levels of aquaporin-4 (AQP4) water channels accompanied by significant neurodegeneration. Increased AQP4 has been associated with neuroinflammatory responses including edema, pro-inflammatory cytokine elevations, arachidonic acid release, and oxidative stress. Co-treatment of ethanol-binged slice cultures with docosahexaenoic acid (DHA), an omega-3 fatty acid known to suppress brain damage from other insults, prevents both the AQP4 elevations and the neurodamage. Surmising that AQP4 augmentation is a causative neuroinflammatory component in this model, we are investigating several possibilities to explain the protective actions of the omega-3 fatty acid. Since the worldwide incidence of cognitive dysfunction and dementia from ethanol abuse and alcoholism is not inconsequential, DHA supplementation with chronic alcoholics could emerge to be a rational approach to potentially lessening brain disabilities.

Effect of repetitive daily ethanol intoxication on adult rat brain: significant changes in phospholipase A2 enzyme levels in association with increased PARP-1 indicate neuroinflammatory pathway activation.

Collaborating on studies of subchronic daily intoxication in juvenile and adult rats, we examined whether the repetitive ethanol treatments at these two life stages altered levels of key neuroinflammation-associated proteins-aquaporin-4 (AQP4), certain phospholipase A2 (PLA2) enzymes, PARP-1 and caspase-3-in hippocampus (HC) and entorhinal cortex (EC). Significant changes in the proteins could implicate activation of specific neuroinflammatory signaling pathways in these rats as well as in severely binge-intoxicated adult animals that are reported to incur degeneration of vulnerable neurons in HC and EC. Male Wistar rats, ethanol-intoxicated (3 g/kg i.p.) once daily for 6 days over an 8-day interval beginning at 37 days old and repeated at age 68-75 days, were sacrificed 1 h after the day 75 dose (blood ethanol, 200- 230 mg/dl). Analysis of HC with an immunoblot technique showed that AQP4, Ca(+2)-dependent PLA2 (cPLA2 IVA), phosphorylated (activated) p-cPLA2, cleaved (89 kD) PARP (c-PARP), and caspase-3 levels were significantly elevated over controls, whereas Ca(+2)-independent PLA2 (iPLA2 VIA) was reduced ∼70%; however, cleaved caspase-3 was undetectable. In the EC, AQP4 was unchanged, but cPLA2 and p-cPLA2 were significantly increased while iPLA2 levels were diminished (∼40%) similar to HC, although just outside statistical significance (p = 0.06). In addition, EC levels of PARP-1 and c-PARP were significantly increased. The ethanol-induced activation of cPLA2 in association with reduced iPLA2 mirrors PLA2 changes in reports of neurotrauma and also of dietary omega-3 fatty acid depletion. Furthermore, the robust PARP-1 elevations accompanied by negligible caspase-3 activation indicate that repetitive ethanol intoxication may be potentiating non-apoptotic neurodegenerative processes such as parthanatos. Overall, the repetitive ethanol treatments appeared to instigate previously unappreciated neuroinflammatory pathways in vivo. The data provide insights into mechanisms of binge ethanol abuse that might suggest new therapeutic approaches to counter neurodegeneration and dementia.

Neuroinflammatory pathways in binge alcohol-induced neuronal degeneration: oxidative stress cascade involving aquaporin, brain edema, and phospholipase A2 activation.

Chronic binge alcohol exposure in adult rat models causes neuronal degeneration in the cortex and hippocampus that is not reduced by excitotoxic receptor antagonists, but is prevented by antioxidants. Neuroinflammatory (glial-neuronal) signaling pathways are believed to underlie the oxidative stress and brain damage. Based on our experimental results as well as increased knowledge about the pro-neuroinflammatory potential of glial water channels, we propose that induction of aquaporin-4 can be a critical initiating factor in alcohol's neurotoxic effects, through the instigation of cellular edema-based neuroinflammatory cascades involving increased phospholipase A2 activities, polyunsaturated fatty acid release/membrane depletion, decreased prosurvival signaling, and oxidative stress. A testable scheme for this pathway is presented that incorporates recent findings in the alcohol-brain literature indicating a role for neuroimmune activation (upregulation of NF-kappaB, proinflammatory cytokines, and toll-like receptors). We present the argument that such neuroimmune activation could be associated with or even dependent on increased aquaporin-4 and glial swelling as well.

Ethanol and adult CNS neurodamage: oxidative stress, but possibly not excitotoxicity.

Evidence from experiments with adult rodents chronically treated with ethanol via either repetitive binges or continuous intake/exposure supports the occurrence of brain oxidative stress and, at least in binge intoxication/rat models, its essential causative role in neurodamage. However, pharmacological antagonism experiments reveal that N-methyl-D-aspartate (NMDA) receptor-dependent excitotoxicity is not responsible for adult mammalian brain neurodegeneration caused by repetitive binge ethanol intoxication and withdrawals. Since NMDA receptor antagonists apparently are untested with respect to neuronal death/loss in continuous intake/ingestion rodent models, e.g., ethanol/water or ethanol/liquid diets, it is therefore erroneous to assert, as is often done, that excitotoxicity is an important mechanism for ethanol-induced adult mammalian brain damage. Alternatively, results from several laboratories indicate that neurodegeneration due to chronic binge ethanol exposure/withdrawal may be dependent on redox transcription factor signaling and neuroinflammatory/oxidative stress pathways (increased arachidonic acid mobilization and pro-inflammatory cytokines; decreased anti-inflammatory cytokines) downstream of microglial/astroglial activation and moderate yet significant brain edema.

Moderate alcohol consumption and cognitive risk.

We reviewed 143 papers that described the relationship between moderate drinking of alcohol and some aspect of cognition. Two types of papers were found: (1) those that provided ratios of risk between drinkers and nondrinkers (74 papers in total) and (2) those that, although they did not provide such ratios, allowed cognition in drinkers to be rated as "better," "no different," or "worse" than cognition in nondrinkers (69 papers in total). The history of research on moderate drinking and cognition can be divided into two eras: 1977-1997 and 1998-present. Phase I (1977-1997) was the era of neuropsychological evaluation involving mostly young to middle-aged (18-50 years old) subjects. Although initial studies indicated moderate drinking impaired cognition, many later studies failed to confirm this, instead finding no difference in cognition between drinkers and nondrinkers. Phase II (1998-present) was and is the era of mental status exam evaluation involving mostly older (≥55 years old) subjects. These studies overwhelmingly found that moderate drinking either reduced or had no effect on the risk of dementia or cognitive impairment. When all the ratios of risk from all the studies in phase II providing such ratios are entered into a comprehensive meta-analysis, the average ratio of risk for cognitive risk (dementia or cognitive impairment/decline) associated with moderate "social" (not alcoholic) drinking of alcohol is 0.77, with nondrinkers as the reference group. The benefit of moderate drinking applied to all forms of dementia (dementia unspecified, Alzheimer's disease, and vascular dementia) and to cognitive impairment (low test scores), but no significant benefit against cognitive decline (rate of decline in test scores) was found. Both light and moderate drinking provided a similar benefit, but heavy drinking was associated with nonsignificantly higher cognitive risk for dementia and cognitive impairment. Although the meta-analysis also indicated that wine was better than beer or spirits, this was based on a relatively small number of studies because most studies did not distinguish among these different types of alcohol. Furthermore, a number of the studies that did make the distinction reported no difference among the effects of these different types of alcohol. Therefore, at present this question remains unanswered. Analysis also showed that the presence of the apolipoprotein E epsilon 4 allele eliminated the benefit of moderate drinking. However, this was based on a relatively small number of studies and several other studies have found a beneficial effect of the epsilon e4 allele. Further studies are necessary to settle this question. The benefit of moderate alcohol for cognition was seen in both men and women, although the amount and pattern of drinking is very different between the two sexes. Lastly, the finding of unaffected or significantly reduced cognitive risk in light to moderate drinkers was seen in 14/19 countries for which country-specific ratio data were available, with three of the five remaining countries showing nonsignificant reductions as well. Overall, light to moderate drinking does not appear to impair cognition in younger subjects and actually seems to reduce the risk of dementia and cognitive decline in older subjects.

Neuroprotective preconditioning of rat brain cultures with ethanol: potential transduction by PKC isoforms and focal adhesion kinase upstream of increases in effector heat shock proteins.

Preconditioning rat hippocampal-entorhinocortical (HEC) slice or cerebellar cell cultures with moderate concentrations of ethanol (20-30 mm) neuroprotects against pro-inflammatory proteins such as HIV-1 glycoprotein 120 (gp120) or amyloid-ß. The neuroprotective mechanism of ethanol is unclear, but it conceivably involves sensors→transducers→effectors, analogous to other preconditioning modalities. We initially found that the preconditioning augmented two likely heat shock protein (HSP) 'effectors', HSP70 and HSP27, and that precluding HSP upregulation abolished neuroprotection. Here we examined whether pro-survival kinases are transducers potentially leading to HSP effectors. In cerebellar cultures, protein kinase C (PKC) activity increased modestly after 2 days of 30 mm ethanol and was significantly induced after 6 days, when neuroprotection against gp120 becomes manifest. After 4 and particularly after 6 days of preconditioning, immunoblots showed highly elevated PKCε levels and moderately increased PKCα and PKCδ, accompanied by increased membrane translocation (activation) of these isoforms. Also, at the latter preconditioning duration, focal adhesion kinase (FAK), an important actin-associated kinase, and its Y397-phosphorylated form (p-FAK) were elevated, along with parallel increases in HSP27, S85p-HSP27 and HSP70. Furthermore, while confirming increased HSP27 and HSP70 in HEC slices ethanol-preconditioned for 6 days, we detected elevations in PKC isoforms, FAK, p-FAK and p-HSP27 in these organotypic cultures. Importantly, PKC inhibition with GF109203X suppressed FAK, HSP70 and HSP27 amplification/activation in ethanol-preconditioned cerebellar cultures, indicating that PKC is an upstream transducer of FAK and the HSP effectors. Neuroprotection associated with increases in HSP27/HSP70 from ethanol preconditioning entails upregulation/activation of PKC isoforms and FAK, the latter kinase implicating actin cytoskeletal prosurvival pathways in brain preconditioning.

Moderate ethanol preconditioning of rat brain cultures engenders neuroprotection against dementia-inducing neuroinflammatory proteins: possible signaling mechanisms.

There is no question that chronic alcohol (ethanol) abuse, a leading worldwide problem, causes neuronal dysfunction and brain damage. However, various epidemiologic studies in recent years have indicated that in comparisons with abstainers or never-drinkers, light/moderate alcohol consumers have lower risks of age-dependent cognitive decline and/or dementia, including Alzheimer's disease (AD). Such reduced risks have been variously attributed to favorable circulatory and/or cerebrovascular effects of moderate ethanol intake, but they could also involve ethanol "preconditioning" phenomena in brain glia and neurons. Here we summarize our experimental studies showing that moderate ethanol preconditioning (MEP; 20-30 mM ethanol) of rat brain cultures prevents neurodegeneration due to beta-amyloid, an important protein implicated in AD, and to other neuroinflammatory proteins such as gp120, the human immunodeficiency virus 1 envelope protein linked to AIDS dementia. The MEP neuroprotection is associated with suppression of neurotoxic protein-evoked initial increases in [Ca(+2)](i) and proinflammatory mediators--e.g., superoxide anion, arachidonic acid, and glutamate. Applying a sensor --> transducer --> effector model to MEP, we find that onset of neuroprotection correlates temporally with elevations in "effector" heat shock proteins (HSP70, HSP27, and phospho-HSP27). The effector status of HSPs is supported by the fact that inhibiting HSP elevations due to MEP largely restores gp120-induced superoxide potentiation and subsequent neurotoxicity. As upstream mediators, synaptic N-methyl-d-aspartate receptors may be initial prosurvival sensors of ethanol, and protein kinase C epsilon and focal adhesion kinase are likely transducers during MEP that are essential for protective HSP elevations. Regarding human consumption, we speculate that moderate ethanol intake might counter incipient cognitive deterioration during advanced aging or AD by exerting preconditioning-like suppression of ongoing neuroinflammation related to amyloidogenic protein accumulation.

Essential involvement of the NMDA receptor in ethanol preconditioning-dependent neuroprotection from amyloid-betain vitro.

In several epidemiological studies, moderate ethanol consumption has been associated with reduced risks of cognitive decline or Alzheimer's dementia. Of potential relevance is that brain cultures preconditioned with moderate ethanol concentrations are resistant to neurotoxic Alzheimer's amyloid-beta (Abeta) peptides. Using rat cerebellar mixed cultures we investigated whether certain membrane receptors were early 'sensors' in moderate ethanol preconditioning (MEP). In a 6-day MEP protocol (30 mM ethanol), neuroprotection from Abeta25-35 was undiminished by antagonism during the first 3 days of either adenosine A(1) or Galpha(i/o) protein-coupled receptors. However, similar cotreatment with memantine or DL-2-amino-5-phosphono-pentanoic acid (AP-5), antagonists of NMDA receptors (NMDAR), abolished neuroprotection, indicating key early involvement of this ionotropic glutamate receptor. Also in these cultures, directly activating NMDAR using subexcitotoxic NMDA preconditioning prevented Abeta neurotoxicity. By day 2 of MEP, we observed increased levels of NMDAR subunits NR1, NR2B, and NR2C that persisted through day 6. Interestingly, memantine co-exposure blocked elevations in the obligatory NR1 subunit. Furthermore, 2 days of MEP significantly increased two indicators of synaptic NMDAR localization, NR2B phospho-Tyr1472, and post-synaptic density 95 scaffolding protein. The results indicate that ethanol preconditioning-dependent neuroprotection is associated with early increases in NR subunits concomitant with enhancement of synaptic localization and activity of NMDAR.

Linking binge alcohol-induced neurodamage to brain edema and potential aquaporin-4 upregulation: evidence in rat organotypic brain slice cultures and in vivo.

Brain edema and derived oxidative stress potentially are critical events in the hippocampal-entorhinal cortical (HEC) neurodegeneration caused by binge alcohol (ethanol) intoxication and withdrawal in adult rats. Edema's role is based on findings that furosemide diuretic antagonizes binge alcohol-dependent brain overhydration and neurodamage in vivo and in rat organotypic HEC slice cultures. However, evidence that furosemide has significant antioxidant potential and knowledge that alcohol can cause oxidative stress through non-edemic pathways has placed edema's role in question. We therefore studied three other diuretics and a related non-diuretic that, according to our oxygen radical antioxidant capacity (ORAC) assays or the literature, possess minimal antioxidant potential. Acetazolamide (ATZ), a carbonic anhydrase inhibitor/diuretic with negligible ORAC effectiveness and, interestingly, an aquaporin-4 (AQP4) water channel inhibitor, prevented alcohol-dependent tissue edema and neurodegeneration in HEC slice cultures. Likewise, in binge alcohol-intoxicated rats, ATZ suppressed brain edema while inhibiting neurodegeneration. Torasemide, a loop diuretic lacking furosemide's ORAC capability, also prevented alcohol-induced neurodamage in HEC slice cultures. However, bumetanide (BUM), a diuretic blocker of Na(+)-K(+)-2Cl(-) channels, and L-644, 711, a nondiuretic anion channel inhibitor--both lacking antioxidant capabilities as well as reportedly ineffective against alcohol-dependent brain damage in vivo--reduced neither alcohol-induced neurotoxicity nor (with BUM) edema in HEC slices. Because an AQP4 blocker (ATZ) was neuroprotective, AQP4 expression in the HEC slices was examined and found to be elevated by binge alcohol. The results further indicate that binge ethanol-induced brain edema/swelling, potentially associated with AQP4 upregulation, may be important in consequent neurodegeneration that could derive from neuroinflammatory processes, for example, membrane arachidonic acid mobilization and associated oxidative stress.

Alcohol in moderation, cardioprotection, and neuroprotection: epidemiological considerations and mechanistic studies.

In contrast to many years of important research and clinical attention to the pathological effects of alcohol (ethanol) abuse, the past several decades have seen the publication of a number of peer-reviewed studies indicating the beneficial effects of light-moderate, nonbinge consumption of varied alcoholic beverages, as well as experimental demonstrations that moderate alcohol exposure can initiate typically cytoprotective mechanisms. A considerable body of epidemiology associates moderate alcohol consumption with significantly reduced risks of coronary heart disease and, albeit currently a less robust relationship, cerebrovascular (ischemic) stroke. Experimental studies with experimental rodent models and cultures (cardiac myocytes, endothelial cells) indicate that moderate alcohol exposure can promote anti-inflammatory processes involving adenosine receptors, protein kinase C (PKC), nitric oxide synthase, heat shock proteins, and others which could underlie cardioprotection. Also, brain functional comparisons between older moderate alcohol consumers and nondrinkers have received more recent epidemiological study. In over half of nearly 45 reports since the early 1990s, significantly reduced risks of cognitive loss or dementia in moderate, nonbinge consumers of alcohol (wine, beer, liquor) have been observed, whereas increased risk has been seen only in a few studies. Physiological explanations for the apparent CNS benefits of moderate consumption have invoked alcohol's cardiovascular and/or hematological effects, but there is also experimental evidence that moderate alcohol levels can exert direct "neuroprotective" actions-pertinent are several studies in vivo and rat brain organotypic cultures, in which antecedent or preconditioning exposure to moderate alcohol neuroprotects against ischemia, endotoxin, beta-amyloid, a toxic protein intimately associated with Alzheimer's, or gp120, the neuroinflammatory HIV-1 envelope protein. The alcohol-dependent neuroprotected state appears linked to activation of signal transduction processes potentially involving reactive oxygen species, several key protein kinases, and increased heat shock proteins. Thus to a certain extent, moderate alcohol exposure appears to trigger analogous mild stress-associated, anti-inflammatory mechanisms in the heart, vasculature, and brain that tend to promote cellular survival pathways.

Binge ethanol-induced neurodegeneration in rat organotypic brain slice cultures: effects of PLA2 inhibitor mepacrine and docosahexaenoic acid (DHA).

Using rat organotypic hippocampal-entorhinal cortical (HEC) slice cultures, we examined whether phospholipase A2 (PLA2) activity is involved in binge alcohol (ethanol)-induced neurodegeneration, and whether docosahexaenoic acid (DHA; 22:6n-3), a fish oil-enriched fatty acid that is anti-inflammatory in brain damage models, is neuroprotective. Assessed with propidium iodide and lactate dehydrogenase (LDH) leakage, neurodamage from ethanol (6 days 100 mM ethanol with four withdrawal periods) was prevented by the PLA2 pan-inhibitor, mepacrine. Also, ethanol-dependent neurodegeneration-particularly in the entorhinal region-was significantly ameliorated by DHA supplementation (25 microM); however, adrenic acid, a 22:4n-6 analog, was ineffective. Consistent with PLA2 activation, [(3)H] liberation was approximately fivefold greater in [(3)H]arachidonic acid-preloaded HEC slice cultures during ethanol withdrawal compared to controls, and DHA supplementation suppressed [(3)H] release to control levels. DHA might antagonize PLA2 activity directly or suppress upstream activators (e.g., oxidative stress); however, other DHA mechanisms could be important in subdueing ethanol-induced PLA2-dependent and independent neuroinflammatory processes.

Visual cortical plasticity following unilateral sensorimotor cortical lesions in the neonatal rat.

Previous work has shown that unilateral sensorimotor cortex (SMC) lesions in newborn rats resulted in an apparent shift of the motor cortex map in the spared hemisphere, particularly of the hindlimb cortex. In view of such findings, the present study was initiated to determine if the visual cortex located both ipsilateral and contralateral to neonatal SMC, or contralateral to occipital cortical (OC) lesions, would show similar remodeling. Visual evoked potentials (VEPs) were used to map the visual cortex electrophysiologically. The results show an expansion of the visual cortex, in both the contralateral and ipsilateral hemisphere, into normally motor cortical areas in adult animals that had sustained unilateral neonatal unilateral SMC lesions. In contrast, similar changes were not seen within the spared visual cortex after unilateral occipital cortical lesions, suggesting that the shift in the visual map was specifically in response to the SMC lesion and was not a generalized response to neonatal cortical damage. Histological analysis showed a functional expansion in the rostral boundary of visual cortex with no corresponding cytoarchitectural alterations.