Chronic alcohol exposure during critical developmental periods differentially impacts persistence of deficits in cognitive flexibility and related circuitry.

Cognitive flexibility in decision making depends on prefrontal cortical function and is used by individuals to adapt to environmental changes in circumstances. Cognitive flexibility can be measured in the laboratory using a variety of discrete, translational tasks, including those that involve reversal learning and/or set-shifting ability. Distinct components of flexible behavior rely upon overlapping brain circuits, including different prefrontal substructures that have separable impacts on decision making. Cognitive flexibility is impaired after chronic alcohol exposure, particularly during development when the brain undergoes rapid maturation. This review examines how cognitive flexibility, as indexed by reversal and set-shifting tasks, is impacted by chronic alcohol exposure in adulthood, adolescent, and prenatal periods in humans and animal models. We also discuss areas for future study, including mechanisms that may contribute to the persistence of cognitive deficits after developmental alcohol exposure and the compacting consequences from exposure across multiple critical periods.

Excipient exposure in very low birth weight preterm neonates.

OBJECTIVE: The excipients benzyl alcohol, propylene glycol and ethanol are present in medications used in the neonatal intensive care unit. Exposure to high levels can have adverse effects in a neonatal population. The objective was to quantify excipient exposure in very low birth weight (VLBW) neonates and identify risk factors associated with greater exposure. STUDY DESIGN: A retrospective record review of VLBW infants admitted over 1 year. Excipient exposures were calculated and multivariable regression analyses identified risk factors for increasing exposure. RESULTS: In total, 98% of subjects were exposed to at least one excipient. A total of 5 to 9% received doses higher than recommended for adults. Necrotizing enterocolitis, seizure, bronchopulmonary dysplasia and longer stay predicted higher excipient exposure. CONCLUSION: The excipients examined are in medications commonly prescribed for VLBW neonates, and cumulative doses may exceed recommended exposures for adults. Although safety profiles have not been established, judicious use of medication containing these excipients is warranted for this population.

Targeting cancer stem-like cells in glioblastoma and colorectal cancer through metabolic pathways.

Cancer stem-like cells (CSCs) are thought to be the main cause of tumor occurrence, progression and therapeutic resistance. Strong research efforts in the last decade have led to the development of several tailored approaches to target CSCs with some very promising clinical trials underway; however, until now no anti-CSC therapy has been approved for clinical use. Given the recent improvement in our understanding of how onco-proteins can manipulate cellular metabolic networks to promote tumorigenesis, cancer metabolism research may well lead to innovative strategies to identify novel regulators and downstream mediators of CSC maintenance. Interfering with distinct stages of CSC-associated metabolics may elucidate novel, more efficient strategies to target this highly malignant cell population. Here recent discoveries regarding the metabolic properties attributed to CSCs in glioblastoma (GBM) and malignant colorectal cancer (CRC) were summarized. The association between stem cell markers, the response to hypoxia and other environmental stresses including therapeutic insults as well as developmentally conserved signaling pathways with alterations in cellular bioenergetic networks were also discussed. The recent developments in metabolic imaging to identify CSCs were also summarized. This summary should comprehensively update basic and clinical scientists on the metabolic traits of CSCs in GBM and malignant CRC.

Role of neurotrophins on postnatal neurogenesis in the thalamus: prenatal exposure to ethanol.

A second wave of neuronal generation occurs in the ventrobasal nucleus of the rat thalamus (VB) during the first three postnatal weeks. The present study tested the hypotheses (1) that postnatal neurogenesis in the VB is neurotrophin-regulated and (2) that ethanol-induced changes in this proliferation are mediated by neurotrophins. The first studies examined the effects of neurotrophins on the numbers of cycling cells in ex vivo preparations of the VB from 3-day-old rats. The proportion of cycling (Ki-67-positive) VB cells was higher in cultured thalamic slices treated with neurotrophins than in controls. Interestingly, this increase occurred with nerve growth factor (NGF) alone or with a combination of NGF and brain-derived neurotrophic factor (BDNF), but not with BDNF alone. Based on these data, the VBs from young offspring of pregnant rats fed an ethanol-containing or an isocaloric non-alcoholic liquid diet were examined between postnatal day (P) 1 and P31. Studies used enzyme-linked immunosorbent assays and immunoblots to explore the effects of ethanol on the expression of neurotrophins, their receptors, and representative signaling proteins. Ethanol altered the expression of neurotrophins and receptors throughout the first postnatal month. Expression of NGF increased, but there was no change in the expression of BDNF. The high affinity receptors (TrkA and TrkB) were unchanged but ethanol decreased expression of the low affinity receptor, p75. One downstream signaling protein, extracellular signal-regulated kinase (ERK), decreased but Akt expression was unchanged. Thus, postnatal cell proliferation in the VB of young rat pups is neurotrophin-responsive and is affected by ethanol.

Nerve growth factor neuroprotection of ethanol-induced neuronal death in rat cerebral cortex is age dependent.

Organotypic cultures of rat cortex were used to test the hypotheses that nerve growth factor (NGF) is neuroprotective for immature cortical neurons and that ethanol abolishes this neuroprotection in a developmental stage-dependent manner. Samples were obtained on gestational day (G) 16 or postnatal day (P) 3 and cultured with ethanol (0 or 400 mg/dl) and NGF (0 or 30 ng/ml) for 72 h. Dying neurons were identified as exhibiting terminal nick-end labeling, immunoreactivity for activated caspase 3, or condensed nuclear chromatin. Two cortical compartments were examined in fetal tissue: a superficial, cell-sparse marginal zone (MZ) and a cell-dense cortical plate (CP). At P3, the CP was subdivided into a cell-dense upper cortical plate (UCP) and a less densely packed lower cortical plate (LCP). Neuronal death in the MZ was affected by neither NGF nor ethanol at both ages. In the fetal CP, NGF did not affect the incidence of cell death, but ethanol increased it. Treatment with NGF caused an upregulation of the expression of Neg, a gene known to be affected by NGF and ethanol. NGF did not ameliorate the ethanol-induced death. In pups, ethanol increased the amount of death in the LCP. NGF did protect against this death. Neither ethanol nor NGF altered the incidence of cell death in the UCP. The laminar-dependent neuroprotection did not correlate with expression of NGF receptors or Neg. Thus, NGF can be protective against the neurotoxic effect of ethanol in the neonatal brain. This effect is site selective and time dependent and it targets postmigratory, differentiating neurons.

Functional studies of frataxin.

Mitochondria generate adenosine triphosphate (ATP) but also dangerous reactive oxygen species (ROS). One-electron reduction of dioxygen in the early stages of the electron transport chain yields a superoxide radical that is detoxified by mitochondrial superoxide dismutase to give hydrogen peroxide. The hydroxyl radical is derived from decomposition of hydrogen peroxide via the Fenton reaction, catalyzed by Fe2+ ions. Mitochondria require a constant supply of Fe2+ for heme and iron-sulfur cluster biosyntheses and therefore are particularly susceptible to ROS attack. Two main antioxidant defenses are known in mitochondria: enzymes that catalytically remove ROS, e.g. superoxide dismutase and glutathione peroxidase, and low molecular weight agents that scavenge ROS, including coenzyme Q, glutathione, and vitamins E and C. An effective defensive system, however, should also involve means to control the availability of pro-oxidants such as Fe2+ ions. There is increasing evidence that this function may be carried out by the mitochondrial protein frataxin. Frataxin deficiency is the primary cause of Friedreich's ataxia (FRDA), an autosomal recessive degenerative disease. Frataxin is a highly conserved mitochondrial protein that plays a critical role in iron homeostasis. Respiratory deficits, abnormal cellular iron distribution and increased oxidative damage are associated with frataxin defects in yeast and mouse models of FRDA. The mechanism by which frataxin regulates iron metabolism is unknown. The yeast frataxin homologue (mYfh1p) is activated by Fe(II) in the presence of oxygen and assembles stepwise into a 48-subunit multimer (alpha48) that sequesters >2000 atoms of iron in a ferrihydrite mineral core. Assembly of mYfhlp is driven by two sequential iron oxidation reactions: a fast ferroxidase reaction catalyzed by mYfh1p induces the first assembly step (alpha --> alpha3), followed by a slower autoxidation reaction that promotes the assembly of higher order oligomers yielding alpha48. Depending on the ionic environment, stepwise assembly is associated with the sequestration of < or = 50-75 Fe(II)/subunit. This Fe(II) is initially loosely bound to mYfh1p and can be readily mobilized by chelators or made available to the mitochondrial enzyme ferrochelatase to synthesize heme. However, as iron oxidation and mineralization proceed, Fe(III) becomes progressively inaccessible and a stable iron-protein complex is produced. In conclusion, by coupling iron oxidation with stepwise assembly, frataxin can successively function as an iron chaperon or an iron store. Reduced iron availability and solubility and increased oxidative damage may therefore explain the pathogenesis of FRDA.

Effects of prenatal exposure to ethanol on the expression of bcl-2, bax and caspase 3 in the developing rat cerebral cortex and thalamus.

Prenatal exposure to ethanol causes neuronal death in somatosensory cortex, but apparently not in the ventrobasal nucleus of the thalamus. Effectors such as bcl-2, bax, and caspase 3 can determine whether a neuron survives or dies. We hypothesize that ethanol differentially affects the expression of these proteins in the cortex and thalamus during the periods of naturally occurring and ethanol-induced neuronal death. Pregnant rats were fed ad libitum with an ethanol-containing liquid diet (Et) or pair-fed an isocaloric non-alcoholic diet (Ct). Samples were collected from fetuses (gestational day (G) 16 and G19) and pups (postnatal day (P) 0 through P30) and examined for bcl-2, bax, or caspase 3 expression using a quantitative immunoblotting procedure. Prenatal exposure to ethanol reduced cortical bcl-2 expression, but not bax expression on P6. Hence, the bcl-2/bax ratio was lower in Et-treated rats than in controls. In contrast, thalamic expression of neither bcl-2 nor bax was significantly different in the two groups of rats. Thus, the thalamic bcl-2/bax ratio was unaffected by exposure to ethanol. During the period of naturally occurring neuronal death, the expression of the active (20 kDa) and inactive isoforms (32 kDa) of caspase 3 was altered in the cortices of Et-treated rats, but not in their thalami. Thus, prenatal exposure to ethanol affected the early postnatal expression of death-related proteins in the cortex, but not in the thalamus. These biochemical changes concur with anatomical data on the spatial and temporal selectivity of ethanol toxicity in the developing CNS.

Episodic exposure to ethanol during development differentially affects brainstem nuclei in the macaque.

Neuronal vulnerability to ethanol may be non-specific, i.e., vulnerability may be conferred by the developmental state of the population or by the site of derivation. To address these issues, the effect of developmental exposure to ethanol on three brainstem nuclei; the trigeminal motor (MoV), facial motor (MoVII) and medial superior olivary (MSO) nuclei was determined. MoVII and MSO are generated at the same time and from the same rhombomere, r4. MoV is generated earlier from r2. Macaca nemestrina were exposed to ethanol or a control solution one day per week for six or 24 weeks of gestation. Brainstems of the mature offspring were sectioned and stained. The number of neurons and volume of each nucleus were determined stereologically. Neuron number was lower in MoV and MSO following exposure to ethanol whereas MoVII appeared unaffected. No significant effects of ethanol exposure were seen on the volume and weight of the brainstem, or the volume of the individual nuclei. These findings show that ethanol differentially affects brainstem nuclei in a targeted, rather than non-specific, manner. Furthermore, they show that serious ethanol-induced neurological deficits can be present without gross morphological changes.

Expression of bcl-2, bax, and caspase-3 in the brain of the developing rat.

Naturally occurring neuronal death (NOND) is generally considered to be apoptotic. Apoptosis is an active form of cell death in which the regulation of specific proteins produces anti- or pro-apoptotic signals. Two of the protein families involved in this regulation are the bcl proteins and caspases. A quantitative immunoblotting technique was used to examine the temporal expression of bcl-2, bax, and two isoforms of caspase 3 (an active 20 kDa isoform and the inactive 32 kDa precursor) throughout the developing neuraxis. Long-Evans rat fetuses were collected on gestational day (G) 16 and G19, and pups were harvested on postnatal day (P) 0, P3, P6, P12, P21, and P30. Brains were divided into five segments: cortex, thalamus, midbrain, medulla/pons, and cerebellum. In general, the expression of bax increased and the ratio of bcl-2 expression to bax expression decreased concurrent with published data on the onset of NOND in a given area. The timing of these events was paralleled by an increase in the expression of active caspase 3. Unlike the bcl proteins, caspase 3 expression returned toward fetal levels as the brain matured. The timing of the changes in bcl protein and caspase expression show that both protein families are involved in promoting neuronal death. Reductions in caspase expression (and not bcl-2 and bax expression) are key to ending the period of NOND.

Effects of prenatal exposure to ethanol on systems matching: the number of neurons in the ventrobasal thalamic nucleus of the mature rat.

Following prenatal exposure to ethanol, rats have a 1/3 fewer neurons in the second order (principal sensory nucleus of the trigeminal nerve) and fourth order neurons (somatosensory cortex) of the trigeminal-somatosensory pathway than do controls. Based on the numerical matching hypothesis, we predict that the number of third-order neurons (in the ventrobasal nucleus of the thalamus; VB) also will show a similar effect of prenatal ethanol exposure. Stereological methods were used to determine the total number of neurons in the VB on postnatal day 30. Surprisingly, prenatal exposure to ethanol had no effect on the VB volume or on the number of VB neurons. Thus, prenatal exposure to ethanol induces numerical imbalances within the trigeminal-somatosensory system.

Long-term effect of postnatal alcohol exposure on the number of cells in the neocortex of the rat: a stereological study.

Behavioral and morphological studies suggest that exposure to alcohol during development may cause damage in the neocortex. In this study, rat pups were exposed to alcohol during the brain growth spurt and examined at adulthood to ascertain the long-term effect of alcohol exposure on the neocortex. Four-day-old rat pups were surgically implanted with an intragastric cannula while under ether anesthesia and artificially reared from postnatal day (PN) 4 through PN11. Two of the consecutive 12 daily feeds contained either alcohol (4.5 g/kg; alcohol-exposed) or an isocaloric maltose/dextrin solution (gastrostomy control) from PN4 through PN9. On PN115, animals were perfused intracardially and the brains removed. Unbiased stereological methods were used to determine the neocortical volume, the total number of neurons and glial cells in the entire neocortex and in layer V, and the mean cell volume of neurons or mean nuclear volume of glial cells in layer V. No effect of alcohol was seen in the neuronal population on either cell number or mean cell volume, nor was there any difference in the total number or mean nuclear volume of glial cells in layer V. These findings suggest that neither the entire neocortex nor layer V alone are vulnerable to permanent alcohol-induced cell death.

Differential effects of two bile salts on ion transport characteristics of teleost intestine.

The effects of a dihydroxy and a trihydroxy bile salt on the Na+- and Cl(-)-absorbing, goby posterior intestine are quite different. Taurochenodeoxycholate, a dihydroxy bile salt, increases tissue permeability to Cl-, primarily by opening the paracellular shunt pathway. The trihydroxy bile salt taurocholate lacks these effects and may, in fact, decrease tissue permeability. In light of the general structural similarity of these two molecules, a detergent action is considered unlikely and, instead, a more specific (perhaps receptor-mediated) mechanism is suggested.