Nicotinamide Inhibits Ethanol-Induced Caspase-3 and PARP-1 Over-activation and Subsequent Neurodegeneration in the Developing Mouse Cerebellum.

Fetal alcohol spectrum disorder (FASD) is the principal preventable cause of mental retardation in the western countries resulting from alcohol exposure during pregnancy. Ethanol-induced massive neuronal cell death occurs mainly in immature neurons during the brain growth spurt period. The cerebellum is one of the brain areas that are most sensitive to ethanol neurotoxicity. Currently, there is no effective treatment that targets the causes of these disorders and efficient treatments to counteract or reverse FASD are desirable. In this study, we investigated the effects of nicotinamide on ethanol-induced neuronal cell death in the developing cerebellum. Subcutaneous administration of ethanol in postnatal 4-day-old mice induced an over-activation of caspase-3 and PARP-1 followed by a massive neurodegeneration in the developing cerebellum. Interestingly, treatment with nicotinamide, immediately or 2 h after ethanol exposure, diminished caspase-3 and PARP-1 over-activation and reduced ethanol-induced neurodegeneration. Conversely, treatment with 3-aminobenzadine, a specific PARP-1 inhibitor, was able to completely block PARP-1 activation, but not caspase-3 activation or ethanol-induced neurodegeneration in the developing cerebellum. Our results showed that nicotinamide reduces ethanol-induced neuronal cell death and inhibits both caspase-3 and PARP-1 alcohol-induced activation in the developing cerebellum, suggesting that nicotinamide might be a promising and safe neuroprotective agent for treating FASD and other neurodegenerative disorders in the developing brain that shares similar cell death pathways.

A proposal for a coordinated effort for the determination of brainwide neuroanatomical connectivity in model organisms at a mesoscopic scale.

In this era of complete genomes, our knowledge of neuroanatomical circuitry remains surprisingly sparse. Such knowledge is critical, however, for both basic and clinical research into brain function. Here we advocate for a concerted effort to fill this gap, through systematic, experimental mapping of neural circuits at a mesoscopic scale of resolution suitable for comprehensive, brainwide coverage, using injections of tracers or viral vectors. We detail the scientific and medical rationale and briefly review existing knowledge and experimental techniques. We define a set of desiderata, including brainwide coverage; validated and extensible experimental techniques suitable for standardization and automation; centralized, open-access data repository; compatibility with existing resources; and tractability with current informatics technology. We discuss a hypothetical but tractable plan for mouse, additional efforts for the macaque, and technique development for human. We estimate that the mouse connectivity project could be completed within five years with a comparatively modest budget.

Early Postnatal Ethanol Exposure in Mice Induces Sex-Dependent Memory Impairment and Reduction of Hippocampal NMDA-R2B Expression in Adulthood.

Drinking alcohol during pregnancy is particularly detrimental for the developing brain and may cause a broad spectrum of cognitive and behavioral impairments, collectively known as fetal alcohol spectrum disorder (FASD). While behavioral abnormalities and brain damage have been widely investigated in animal models of FASD, the sex differences in the vulnerability to perinatal ethanol exposure have received less consideration. Here we investigated the long-term behavioral and molecular effects of acute ethanol-binge like exposure during the early postnatal period (equivalent to the third trimester of human pregnancy) in adult male and female mice. CD1 mice received a single ethanol exposure on P7 and were analyzed starting from P60. We found that ethanol-exposed mice showed increased activity in the open field test and in the plus-maze test, regardless of the sex. Interestingly, only ethanol-exposed adult male mice exhibited memory impairment in the water maze and fear-conditioning tests. Remarkably, hippocampal levels of NMDA-R2B were reduced only in ethanol-exposed male, while total BDNF levels were increased in both male and female ethanol-exposed mice. Our data suggest a different susceptibility of early postnatal ethanol exposure in male and female CD1 mice.

Alcohol regulates gene expression in neurons via activation of heat shock factor 1.

Drinking alcohol causes widespread alterations in gene expression that can result in long-term physiological changes. Although many alcohol-responsive genes (ARGs) have been identified, the mechanisms by which alcohol alters transcription are not well understood. To elucidate these mechanisms, we investigated Gabra4, a neuron-specific gene that is rapidly and robustly activated by alcohol (10-60 mM), both in vitro and in vivo. Here we show that alcohol can activate elements of the heat shock pathway in mouse cortical neurons to enhance the expression of Gabra4 and other ARGs. The activation of Gabra4 by alcohol or high temperature is dependent on the binding of heat shock factor 1 (HSF1) to a short downstream DNA sequence, the alcohol response element (ARE). Alcohol and heat stimulate the translocation of HSF1 from the cytoplasm to the nucleus and the induction of HSF1-dependent genes, Hsp70 and Hsp90, in cultured neurons and in the mouse cerebral cortex in vivo. The reduction of HSF1 levels using small interfering RNA prevented the stimulation of Gabra4 and Hsp70 by alcohol and heat shock. Microarray analysis showed that many ARGs contain ARE-like sequences and that some of these genes are also activated by heat shock. We suggest that alcohol activates phylogenetically conserved pathways that involve intermediates in the heat shock cascade and that sequence elements similar to the ARE may mediate some of the changes in gene expression triggered by alcohol intake, which could be important in a variety of pathophysiological responses to alcohol.

Sortilin controls intracellular sorting of brain-derived neurotrophic factor to the regulated secretory pathway.

Brain-derived neurotrophic factor (BDNF), after activity-dependent secretion from neurons, modulates critical nervous system functions. Recently, a variant in the human bdnf gene, resulting in a valine to methionine substitution in the prodomain, has been shown to lead to defective regulated secretion from neurons and memory impairment. Here, we report a novel function for a Vps10p domain protein, sortilin, in controlling BDNF sorting to the regulated secretory pathway. Sortilin interacts specifically with BDNF in a region encompassing the methionine substitution and colocalizes with BDNF in secretory granules in neurons. A truncated form of sortilin causes BDNF missorting to the constitutive secretory pathway without affecting neurotrophin-4 (NT-4) secretion. In addition, sortilin small interfering RNA introduced into primary neurons also led to BDNF missorting from the regulated to the constitutive secretory pathway. Together, these data suggest a mechanism to understand the defect associated with variant BDNF and provide a framework, based on divergent presynaptic regulation of sorting to secretory pathways, to explain how two ligands for tropomyosin-related kinase B, BDNF and NT-4, can mediate diverse biological responses.

Nicotinamide protects against ethanol-induced apoptotic neurodegeneration in the developing mouse brain.

BACKGROUND: Exposure to alcohol during brain development may cause a neurological syndrome called fetal alcohol syndrome (FAS). Ethanol induces apoptotic neuronal death at specific developmental stages, particularly during the brain-growth spurt, which occurs from the beginning of third trimester of gestation and continues for several years after birth in humans, whilst occurring in the first two postnatal weeks in mice. Administration of a single dose of ethanol in 7-d postnatal (P7) mice triggers activation of caspase-3 and widespread apoptotic neuronal death in the forebrain, providing a possible explanation for the microencephaly observed in human FAS. The present study was aimed at determining whether nicotinamide may prevent ethanol-induced neurodegeneration. METHODS AND FINDINGS: P7 mice were treated with a single dose of ethanol (5 g/kg), and nicotinamide was administered from 0 h to 8 h after ethanol exposure. The effects of nicotinamide on ethanol-induced activation of caspase-3 and release of cytochrome-c from the mitochondria were analyzed by Western blot (n = 4-7/group). Density of Fluoro-Jade B-positive cells and NeuN-positive cells was determined in the cingulated cortex, CA1 region of the hippocampus, and lateral dorsal nucleus of the thalamus (n = 5-6/group). Open field, plus maze, and fear conditioning tests were used to study the behavior in adult mice (n = 31-34/group). Nicotinamide reduced the activation of caspase-3 (85.14 +/- 4.1%) and the release of cytochrome-c (80.78 +/- 4.39%) in postnatal mouse forebrain, too. Nicotinamide prevented also the ethanol-induced increase of apoptosis. We demonstrated that ethanol-exposed mice showed impaired performance in the fear conditioning test and increased activity in the open field and in the plus maze. Administration of nicotinamide prevented all these behavioral abnormalities in ethanol-exposed mice. CONCLUSIONS: Our findings indicate that nicotinamide can prevent some of the deleterious effects of ethanol on the developing mouse brain when given shortly after ethanol exposure. These results suggest that nicotinamide, which has been used in humans for the treatment of diabetes and bullous pemphigoid, may hold promise as a preventive therapy of FAS.

Multipotent neural stem cells reside into the rostral extension and olfactory bulb of adult rodents.

The lateral walls of the forebrain lateral ventricles are the richest source of stem cells in the adult mammalian brain. These stem cells give rise to new olfactory neurons that are renewed throughout life. The neurons originate in the subventricular zone (SVZ), migrate within the rostral extension (RE) of the SVZ along the rostral migratory stream (RMS) within tube-like structures formed of glial cells, to eventually reach the olfactory bulb (OB). We demonstrate that, contrary to the current view, multipotential (neuronal-astroglial-oligodendroglial) precursors with stem cell features can be isolated not only from the SVZ but also from the entire RE, including the distal portion within the OB. Specifically, these stem cells do not derive from the migratory neuroblasts coming from the SVZ. Interestingly, stem cells isolated from the proximal RE generate significantly more oligodendrocytes, and those from the distal RE proliferate significantly more slowly than stem cells derived from the SVZ and other RE regions. These findings demonstrate that stem cells are not confined to the forebrain periventricular region and indicate that stem cells endowed with different functional characteristics occur at different levels of the SVZ-RE pathway.

Investigating the use of primary adult subventricular zone neural precursor cells for neuronal replacement therapies.

With the relatively recent discovery that neurogenesis persists throughout life in restricted regions of the adult mammalian brain, including those of human beings, there has been great interest in the use of adult-derived neural stem cells for neuronal replacement. There are many great hurdles that must be overcome in order for such replacement strategies to succeed. In this review, we outline some of these hurdles and discuss recent experiments that investigate the potential of using neural precursor cells found in the subventricular zone of the adult brain for brain repair.

Functional reduction of SK3-mediated currents precedes AMPA-receptor-mediated excitotoxicity in dopaminergic neurons.

In primary cultures of mesencephalon small-conductance calcium-activated potassium channels (SK) are expressed in dopaminergic neurons. We characterized SK-mediated currents (I(SK)) in this system and evaluated their role on homeostasis against excitotoxicity. I(SK) amplitude was reduced by the glutamatergic agonist AMPA through a reduction in SK channel number in the membrane. Blockade of I(SK) for 12 h with apamin or NS8593 reduced the number of dopaminergic neurons in a concentration-dependent manner. The effect of apamin was not additive to AMPA toxicity. On the other hand, two I(SK) agonists, 1-EBIO and CyPPA, caused a significant reduction of spontaneous loss of dopaminergic neurons. 1-EBIO reversed the effects of both AMPA and apamin as well. Thus, I(SK) influences survival and differentiation of dopaminergic neurons in vitro, and is part of protective homeostatic responses, participating in a rapidly acting negative feedback loop coupling calcium levels, neuron excitability and cellular defenses. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Single alcohol exposure in early life damages hippocampal stem/progenitor cells and reduces adult neurogenesis.

Alcohol exposure during pregnancy may cause fetal alcohol syndrome (FAS), characterized by impaired cognitive functions. Neurogenesis occurs in the adult hippocampus and is functionally associated with learning, memory, and mood disorders. However, whether early postnatal exposure to alcohol impairs neurogenesis and through which mechanisms it occurs is poorly understood. Here, we report that a single episode of alcohol exposure in postnatal day 7 (P7) decreases neurogenesis in the adult hippocampus. Furthermore, we demonstrate a co-localization of glial fibrillar acidic protein, nestin, and vimentin with activated caspase-3 12 h after ethanol treatment. Finally, we show that the number of primary neurospheres derived from the hippocampi of alcohol-exposed mice is reduced compared to controls. These findings suggest that alcohol exposure in postnatal mice reduces the pool of neural stem/progenitor cells in the DG, and subsequently results in a decrease of adult neurogenesis. This may explain certain aspects of impaired hippocampal functions in FAS.

Cognitive enhancement with central thalamic electrical stimulation.

Central thalamic electrical stimulation has been proposed as a method for remediation of acquired cognitive disability. Long-standing experimental and clinical observations indicate a key role for neurons within the central thalamus in maintaining the alert waking state and facilitating attended behaviors. Here, we show that continuous high frequency (100 Hz) electrical stimulation of the central thalamus generates widespread cortical activation of c-fos across all cortical layers and a selective pattern of regulation of zif268 within the supragranular, granular, and infragranular cortical laminae. Significant elevation of both immediate early genes also is seen in the dentate gyrus of the hippocampus. Use of the same stimulation parameters is shown to facilitate untrained goal-directed seeking behavior and object recognition memory in rodents. An overall increase of exploratory motor behaviors and grooming activity also is observed, consistent with a global increase in arousal. Taken together, these studies indicate that electrical stimulation of the central thalamus may enhance cognitive performance through neocortical and hippocampal neuronal activation and specific regulation of gene expression.

Genetic variant BDNF (Val66Met) polymorphism alters anxiety-related behavior.

A common single-nucleotide polymorphism in the brain-derived neurotrophic factor (BDNF) gene, a methionine (Met) substitution for valine (Val) at codon 66 (Val66Met), is associated with alterations in brain anatomy and memory, but its relevance to clinical disorders is unclear. We generated a variant BDNF mouse (BDNF(Met/Met)) that reproduces the phenotypic hallmarks in humans with the variant allele. BDNF(Met) was expressed in brain at normal levels, but its secretion from neurons was defective. When placed in stressful settings, BDNF(Met/Met) mice exhibited increased anxiety-related behaviors that were not normalized by the antidepressant, fluoxetine. A variant BDNF may thus play a key role in genetic predispositions to anxiety and depressive disorders.

Repetitive hypoglycemia in young rats impairs hippocampal long-term potentiation.

Mechanisms underlying cognitive dysfunction in young diabetic children are poorly understood, and may include synaptic dysfunction from insulin-induced hypoglycemia. We developed a model of repetitive insulin-induced hypoglycemia in young rats and examined hippocampal long-term potentiation, an electrophysiologic assay of synaptic plasticity, 3-5 d after the last hypoglycemic event. Three hypoglycemic events between postnatal d 21-25 produced modest cortical (17 +/- 2.9 dead neurons per section in parasagittal cortex), but not hippocampal, neuron death quantified by Fluoro-Jade B staining. There was no change in neurogenesis in the hippocampal dentate granule cell region by quantification of bromodeoxyuridine incorporation. Although normal baseline hippocampal synaptic responses were elicited from hippocampal slices from hypoglycemic animals, long-term synaptic potentiation could not be induced in hippocampal slices from rats subjected to hypoglycemia. These results suggest that repetitive hypoglycemia in the developing brain can cause selective impairment of synaptic plasticity in the absence of cell death, and without complete disruption of basal synaptic transmission. We speculate that impaired synaptic plasticity in the hippocampus caused by repetitive hypoglycemia could underlie memory and cognitive deficits observed in young diabetic children, and that cortical neuron death caused by repetitive hypoglycemia in the developing brain may contribute to other neurologic, cognitive, and psychological problems sometimes encountered in diabetic children.

Selective impairment of hippocampal neurogenesis by chronic alcoholism: protective effects of an antioxidant.

A major pathogenic mechanism of chronic alcoholism involves oxidative burden to liver and other cell types. We show that adult neurogenesis within the dentate gyrus of the hippocampus is selectively impaired in a rat model of alcoholism, and that it can be completely prevented by the antioxidant ebselen. Rats fed for 6 weeks with a liquid diet containing moderate doses of ethanol had a 66.3% decrease in the number of new neurons and a 227-279% increase in cell death in the dentate gyrus as compared with paired controls. Neurogenesis within the olfactory bulb was not affected by alcohol. Our studies indicate that alcohol abuse, even for a short duration, results in the death of newly formed neurons within the adult brain and that the underlying mechanism is related to oxidative or nitrosative stress. Moreover, these findings suggest that the impaired neurogenesis may be a mechanism mediating cognitive deficits observed in alcoholism.