Brain myelination in the offspring of ethanol-treated rats: in Utero versus lactational exposure by crossfostering offspring of control, pairfed and ethanol treated dams

Pregnant Long-Evans rats were received on day 5 of gestation and divided into 4 treatment groups: (1) 27% calories provided as ethanol in a liquid diet; (2) pairfed i.e., isocaloric liquid diet restricted to match group (1); (3) liquid diet provided ad libitum; and (4) laboratory chow and water provided ad libitum. Litters were culled to 8 pups at birth and crossfostered across dams in all 4 groups to provide offspring falling into 16 different experimental groups, including some exposed to ethanol in utero only and some exposed only during lactation. At birth, blood alcohol levels of dams, culled pups and alcohol levels in the stomach contents of culled pups were measured. All pups were weaned and fed laboratory chow and water ad libitum from 21 days onward. At ages 16, 21, 30 and 52 days, pups were sacrificed, and organ/body weight ratios and brain myelin concentrations were determined. Ethanol treated dams had longer gestational periods. The offspring of ethanol treated dams which were crossfostered to pairfed and well nourished dams during lactation had delayed eye opening, persistent lag in body growth and slightly lower brain myelin concentrations. Offspring of dams which were either pairfed or well nourished during gestation, but crossfostered during lactation to ethanol treated dams, had abnormal organ weights, abnormal brain weights and severely depressed brain myelin concentrations persisting through 52 days of age. Thus, lactational ethanol effects on brain myelin were more severe than gestational effects; body growth was affected more severely by gestational exposure, and gestational effects were generally less severe with adequate nutrition.     

Glycoproteins and proteins in an axolemma-enriched fraction and myelin from developing rats: effect of maternal ethanol consumption

The present study examined proteins and glycoproteins from an axolemma-enriched fraction from the developing offspring of female rats that were pair-fed control or 6.6% (50 g/liter) ethanol liquid diets on a chronic basis prior to parturition. In addition, this study examined the synthesis of the major CNS myelin-associated glycoprotein (MAG) as an index of myelin maturation. The results of the latter study demonstrated normal MAG maturation in ethanol-treated rats. However, a significant decrease in the proportion of radioactivity associated with MAG was found in the developing offspring of ethanol-treated rats. The major axolemmal proteins from 32-day rats included those with molecular weights of 105 K, 81 K, 62 K, 55 K, 52 K, 36 K, and 33 K. Major peaks of radioactivity were associated with fucosylated axolemmal glycoproteins with apparent molecular weights of 150 K, 130 K, 85 K, 76 K, and 64 K. Several development-related changes in the protein composition of axolemma-enriched fractions were observed in control animals. Between 22 and 32 days of age control rats exhibited a significant (P less than .05) decrease in the proportion of axolemmal proteins that had apparent molecular weights of 150 K, 105 K, and 62 K. A development-related decrease in the 105 K axolemma-associated protein did not occur during the 22-32 day age period in the offspring of ethanol-treated animals. At 22 days the proportion of this 105 K protein in affected offspring was significantly (P less than .05) less than that in age-matched control rats and comparable to that in 32-day control rats. The relative distribution of radioactivity among fucosylated axolemmal glycoproteins also changed significantly between 22 and 32 days of age. These changes include a decrease in the proportion of radioactivity associated with the 110 K, 55 K, and 52 K fucosylated glycoproteins and an increase in the proportion of radioactivity associated with the 85 K and 67 K glycoproteins. Several small, but significant (P less than .05) alterations were found associated with glycoproteins in an axolemma-enriched fraction from 22- and 32-day ethanol-treated rats.     

The relationship between nutritional adequacy and brain myelin accumulation: a comparison of varying degrees of well fed and undernourished rats

Rats undernourished through 17 days of age to produce mild weight lag (less than 20%) had either the same amount, or slightly more myelin than well fed controls. Schedules of undernourishment producing approximately 30, 40 and 50% body weight lags produced corresponding rank-ordered myelin deficits of 25, 55 and 60%, respectively. Brain growth was relatively spared in all case4s, never exceeding a deficit of 10%. Absolute myelin deficits did not recover following nutritional rehabilitation, although myelin continued to increase in both normal and all test populations.     

Morphine Analgesia is potentiated in adult rats prenatally exposed to ethanol

Exposure to inescapable, intermittent footshock elicits an opioid-mediated stress-induced analgesia in rats. We have previously shown that this response is markedly potentiated in adult rats, prenatally exposed to ethanol. To further investigate our hypothesis that endogenous opioid pain-inhibitory systems are modified by prenatal ethanol exposure, we have measured the analgesic response to morphine, in vitro brain opiate receptor binding characteristics, and occupation of brain opiate receptors following systemic administration of morphine. Compared to controls, rats prenatally exposed to ethanol had significantly enhanced morphine analgesia. This enhancement, however, does not appear attributable to changes in number or affinity of mu or delta opiate receptors, or to altered occupation of receptors by morphine.     

Cyanidin-3-glucoside ameliorates ethanol neurotoxicity in the developing brain

Ethanol exposure induces neurodegeneration in the developing central nervous system (CNS). Fetal alcohol spectrum disorders (FASD) are caused by ethanol exposure during pregnancy and are the most common nonhereditary cause of mental retardation. It is important to identify agents that provide neuroprotection against ethanol neurotoxicity. Multiple mechanisms have been proposed for ethanol-induced neurodegeneration, and oxidative stress is one of the most important mechanisms. Recent evidence indicates that glycogen synthase kinase 3β (GSK3β) is a potential mediator of ethanol-mediated neuronal death. Cyanidin-3-glucoside (C3G), a member of the anthocyanin family, is a potent natural antioxidant. Our previous study suggested that C3G inhibited GSK3β activity in neurons. Using a third trimester equivalent mouse model of ethanol exposure, we tested the hypothesis that C3G can ameliorate ethanol-induced neuronal death in the developing brain. Intraperitoneal injection of C3G reduced ethanol-meditated caspase-3 activation, neurodegeneration, and microglial activation in the cerebral cortex of 7-day-old mice. C3G blocked ethanol-mediated GSK3β activation by inducing phosphorylation at serine 9 while reducing the phosphorylation at tyrosine 216. C3G also inhibited ethanol-stimulated expression of malondialdehyde (MDA) and p47phox, indicating that C3G alleviated ethanol-induced oxidative stress. These results provide important insight into the therapeutic potential of C3G.     

The effects of ethanol on the developing cerebellum and eyeblink classical conditioning

In rats, developmental ethanol exposure has been used to model the central nervous system deficits associated with human fetal alcohol syndrome. Binge-like ethanol exposure of neonatal rats depletes cells in the cerebellum, including Purkinje cells, granule cells, and deep nuclear cells, and produces deficits in simple tests of motor coordination. However, the extent to which anatomical damage is related to behavioral deficits has been difficult to estimate. Eyeblink classical conditioning is known to engage a discrete brain stem-cerebellar circuit, making it an ideal test of cerebellar functional integrity after developmental ethanol exposure. Eyeblink conditioning is a simple form of motor learning in which a neutral stimulus (such as a tone) comes to elicit an eyeblink when repeatedly paired with a stimulus that evokes an eyeblink prior to training (such as mild periorbital stimulation). In eyeblink conditioning, one of the deep cerebellar nuclei, the interpositus nucleus, as well as specific Purkinje cell populations, are sites of convergence for tone conditioned stimulus and somatosensory unconditioned stimulus information, and, together with brain stem nuclei, provide the necessary and sufficient substrate for the learned response. A series of studies have shown that eyeblink conditioning is impaired in both weanling and adult rats given binge-like exposure to ethanol as neonates. In addition, interpositus nucleus neurons from ethanol-exposed rats showed impaired activation during eyeblink conditioning. These deficits are accompanied by a permanent reduction In the deep cerebellar nuclear cell population. Because particular cerebellar cell populations are utilized in well-defined ways during eyeblink conditioning, conclusions regarding the underlying neural substrates of behavioral change after developmental ethanol exposure are greatly strengthened.     

Short- and long-term effects of combined pre- and postnatal ethanol exposure (three trimester equivalency) on the development of myelin and axons in rat optic nerve

This study evaluated the effects of a combined gestational and 10 day postnatal alcohol exposure (human three trimester equivalency) on the development of myelin and axons in rat optic nerve. Rats were exposed during gestation via liquid diet, then their artificially reared pups were further exposed for 10 postnatal days via an ethanol-containing diet fed by gastrostomy. Control animals from pair-fed dams were artificially reared for 10 days on pair-fed isocaloric diets. Anesthetized animals were perfused with fixative on gestational days (G) 15 and 20 and postnatal days (P) 5, 10, 15, 20, and 90, then optic nerve tissues prepared for electron microscopy. Optic nerve cross-sectional areas were generally less from G20 through P90 in ethanol exposed animals. Counts of the number of myelinated nerve fibers per unit area and of the numbers of fibers in different stages of myelin development revealed that alcohol exposure caused a delay in myelin acquisition at 10 and 15 days that was compensated for at 20 and 90 days. Myelin thickness as a function of axon diameter was decreased in the alcohol exposed animals from 10 through 90 days, indicating a permanent reduction in the relative thickness of myelin.

These results show that alcohol exposure for all of gestation and 10 postnatal days in the rat (human three trimester equivalency) causes a permanent reduction in myelin thickness along with a delay in myelin acquisition in the optic nerve. Such alterations in developing and adult myelin could help to explain some of the neurological and visual dysfunctions associated with developmental alcohol exposures.     

Inhibition of the synthesis of glycosphingolipids affects the translocation of proteolipid protein to the myelin membrane

Brain slices obtained from young rats were incubated with different radioactive precursors, in the presence and absence of L-cycloserine (an inhibitor of the synthesis of sphingosine) in order to explore the possibility that transport of proteolipids--and specifically of the major myelin proteolipid PLP--to the myelin membrane could be coupled to the transport of cerebrosides or sulfatides. At a concentration of 0.15 mM L-cycloserine, the incorporation of [3H] glycine into total proteins, proteolipid apoproteins (APL), PLP, and myelin basic proteins (MBP) of the total homogenate was unaffected by the presence of the inhibitor, whereas the incorporation of [3H] serine into glycosphingolipids decreased markedly. Under similar incubation conditions, the entry of labeled APL and of PLP into the myelin membranes in the presence of L-cycloserine decreased markedly (50%) in comparison to controls. Entry of MBP was not affected by the inhibitor. These results indicate that when synthesis of glycosphingolipids is inhibited by L-cycloserine, thus decreasing the availability of cerebrosides and sulfatides, the translocation of PLP to myelin is disrupted, suggesting that its transport through the oligodendroglial cell could be coupled to the transport of glycosphingolipids and, most probably, of sulfatides.     

Maternal dietary restriction causes myelin and lipid deficits in the brain of offspring

The relationship between brain myelination and nutritional insufficiency in suckling rats whose dams had a restricted dietary intake was studied. The undernourished pups were characterized by body weight and brain weight that were 40-70% and 82-88% that of controls throughout the suckling period. Myelin concentrations, whether expressed as mg protein or as mg myelin dry weight per g wet brain, were 30-40% of normally fed controls. Myelin of undernourished rats contained total lipid, cholesterol, phospholipids, and sphingolipids that were 42, 54, 39, and 48%, respectively of the control counterparts. There was no change in the mole ratio of cholesterol:phospholipids:sphingolipids (i.e., cerebroside + sulfatide) in myelin from undernourished rats. Concentrations of individual phospholipids and sphingolipids were lowered by approximately the same percentage. Despite long-lasting, irreversible stunting of whole-body and brain growth, concentrations of myelin and myelin lipids returned to control levels after nutritional rehabilitation. Since the observed effects are different from those of more commonly used models, the present form of undernutrition may offer a useful system for studying the relationship between myelin lipids and brain development.     

Maternal ethanol consumption and synaptic membrane glycoproteins in offspring

The present study was undertaken to assess the influences of chronic maternal ethanol consumption, prior to and during gestation, on the development of synaptic plasma membranes (SPMs) and on the synthesis of SPM glycoproteins in offspring. Comparisons were made between animals whose mothers were pair-fed a control or 6.6% (v/v) ethanol liquid diet in which protein accounted for either 18% (original) (C & E) or 21% (revised) (*C & *E) of the calories. In addition, groups of pups that were either cross-fostered (*C & *E) with chow-fed surrogate mothers or reverse cross-fostered (offspring of chow-fed mothers with *C & *E mothers) were examined. Ethanol and matched (same dietary group) control pups had comparable brain and body weights, brain protein content, and yield of SPM proteins during the 10–24 day age period examined. However, the yield of SPM proteins from ethanol and control offspring of and/or reared by the three groups of rat mothers that received the *E and *C liquid diets was greater than that of the offspring of rats that were fed the original diets. This suggests that the original diets were not nutritionally adequate for pregnant rats. Despite the fact that the content of SPM proteins was comparable in ethanol and matched control pups, the offspring of ethanol-treated rats had an abnormal distribution of [³H]-or [¹⁴C]-fucose-derived radioactivity among SPM glycoproteins. The SPM abnormalities were most severe in the non-cross-fostered offspring of E rats. No SPM glycoprotein abnormalities were found in the reverse cross-fostered group. The results of the present study demonstrate that chronic maternal ethanol consumption prior to parturition has a severe effect on the synthesis of SPM glycoproteins in developing offspring without affecting the content of SPMs per se. It also demonstrates the importance of optimizing the composition of liquid diets used to feed pregnant rats.     

Expression of myelin proteins in the developing human spinal cord: cloning and sequencing of human proteolipid protein cDNA

A full-length clone for the human proteolipid protein (PLP) was isolated from a cDNA library constructed from poly(A)+ RNA isolated from fetal spinal cords obtained at 15-24 weeks of conceptional age. The sequence of the human PLP cDNA was determined, and the deduced amino acid sequence was found to be identical with that of rat PLP. Comparison of human and rat PLP cDNA clones indicated that the coding regions retained 97% homology and that there were also other areas of conserved sequence. The human 5'-untranslated region was 93% homologous to that of the rat. The 3'-untranslated region was, overall, 73% homologous to that of the rat with areas containing greater than 84% homology in the first 400 and last 200 nucleotides. The most variability within the 3'-untranslated region occurred between nucleotides 2,000-2,500, where homology with the rat cDNA dropped to 55%. Expression of PLP in the human spinal cord between 11 and 23 weeks after conception was examined and compared with the expression of the myelin basic protein (MBP). RNA was isolated from pooled human spinal cords obtained at three periods of development: 11-14 weeks, 17-19 weeks, and 21-23 weeks. Northern blot analysis revealed a 3.2-kilobase (kb) PLP mRNA that was present at higher abundance in the 21-23-week spinal cord RNA than in the 17-19-week or the 11-14-week samples. The 17-19-week RNA sample also contained a PLP-hybridizing band at 2.2 kb which may possibly have arisen by utilization of alternative polyadenylation signals. Messenger RNA for MBP was detectable at 11-14 weeks but was readily evident in both the 17-19- and 21-23-week age groups. Immunoblot analysis of whole spinal cord homogenates indicated that polypeptides for MBP preceded the appearance polypeptides for PLP by 3-4 weeks.     

Different adaptations in ventral tegmental area dopamine neurons in control and ethanol exposed rats after methylphenidate treatment.

BACKGROUND: Methylphenidate (MPH) is a psychostimulant effective in treating attention-deficit/hyperactivity disorder (ADHD). Repeated MPH treatment may increase substance abuse risk because of adaptations in dopaminergic (DA) function associated with sensitization to subsequent stimulant exposure. However, this possibility is based on observations in normal animals and may not apply to animals with attention problems linked to compromised DA function such as prenatal ethanol exposed (PE) animals. METHODS: The electrical activity of ventral tegmental area (VTA) DA neurons was studied after the cessation of repeated MPH treatment at a threshold dose (1 mg/kg/day for 3 weeks) in PE and control rats. RESULTS: In control rats, there was a continuous increase in VTA DA neuron excitability post-MPH treatment, characterized by a transient increase in population activity (1 day posttreatment) followed by decreased population activity (30-60 days posttreatment) in most of the animals due to depolarization inactivation. In PE rats, MPH treatment decreased the excessive excitability of VTA DA neurons and resulted in prolonged normalization in the population activity (1-60 days posttreatment). These changes were not mediated by altered sensitivity of somatodendritic DA autoreceptors. CONCLUSIONS: Repeated MPH treatment produced distinctly different effects on VTA DA neuron activity in control and PE animals. These results suggest that repeated MPH treatment for ADHD may not lead to increased substance abuse risk in special populations such as individuals with fetal alcohol spectrum disorder.     

Effects of maternal ethanol consumption in rats on basal and rhythmic pituitary - adrenal function in neonatal offspring

Neonatal corticoid treatment delays development of the circadian rhythm of plasma corticosterone in rats. We therefore sought to determine whether fetal or neonatal exposure to ethanol, a substance which activates the hypothalamo - pituitary - adrenal axis, produces similar effects. Subjects were the offspring of dams fed a 5.0% w/v ethanol-containing liquid diet or pair-fed an isocaloric control diet during gestation weeks two and three or during postnatal week one.At birth (day 1), the fetal ethanol-exposed pups had significantly higher brain and plasma corticosterone levels than the pair-fed or normal controls; brain and body weights were unaffected. By day 3, brain and plasma corticosterone titers in the fetal ethanol-exposed pups declined to the levels of the pair-fed and normal controls, although brain weights were significantly reduced. Significantly higher p.m. than a.m. levels of plasma corticosterone first occurred on day 18 both in the fetal ethanol-exposed pups and in the pair-fed and normal controls. Thus, despite its causing elevated corticosterone levels at birth, fetal exposure to ethanol did not affect the onset of the pituitary - adrenal circadian rhythm.On the other hand, exposure to ethanol during the first neonatal week delayed the onset of the pituitary - adrenal rhythm from day 18 to day 21. However, even greater delays occurred in the neonatal pair-fed controls, suggesting that the delays following neonatal exposure were due to nutritional deficits rather than to alcohol per se. The developmental and long-term influences of elevated corticoid levels at birth in fetal ethanol-exposed rats on other aspects of pituitary - adrenal function remain to be determined.     

Attention,locomotor activity and developmental milestones in rats prenatally exposed to ethanol

RationaleDecline of attentional performance as a function of time engaged on a task and hyperactivity are features shared by children and adults with fetal alcohol syndrome or attentional deficit and hyperactivity disorders.ObjectiveTo investigate the effects of prenatal exposure to two doses of ethanol on developmental milestones, locomotor activity and attention.MethodsWistar rats born from dams exposed to one of four maternal treatments during pregnancy were used: A35 – liquid diet with 35% ethanol-derived calories; A10 – liquid diet with 10% ethanol-derived calories; control – ethanol-free liquid diet; chow – laboratory chow and water.ResultsA35 performed worse in grip strength than control and chow (postnatal day – 14, p < 0.05) but not in negative geotaxis (postnatal days 7–10); A35 also showed more locomotor activity than control and A10 (p < 0.05). Regarding attention, acquisition of the five choice reaction time task was similar between groups, but, the percentage of omission errors from A35 group was greater than other groups at baseline parameters, at shorter (2 s) and longer (7 s) inter-trial intervals and at a shorter stimulus duration (0.5 s) (p < 0.05). The percentage of omissions was larger in A35 as the blocks progressed in sessions with either longer or shorter inter-trial intervals (group × block p < 0.05). Animals from A10 group did not show any impairment in the tasks performed.ConclusionsOur study demonstrates that as well as developmental impairments, prenatal ethanol can produce deficits associated with an increase in attentional demand in rodents, analogous to those observed in fetal alcohol syndrome and attentional deficit and hyperactivity disorders.     

Developmental abnormalities in the nerves of peripheral myelin protein 22-deficient mice

Peripheral myelin protein 22 (PMP22) is a tetraspan glycoprotein whose misexpression is associated with a family of hereditary peripheral neuropathies. In a recent report, we have characterized a novel PMP22-deficient mouse model in which the first two coding exons were replaced by the lacZ reporter. To investigate further the myelin abnormalities in the absence of PMP22, sciatic nerves and dorsal root ganglion (DRG) neuron explant cultures from PMP22-deficient mice were studied at various stages of myelination. Throughout the first 3 months of postnatal development, myelin protein and beta4 integrin levels are dramatically reduced, whereas p75 and beta1 integrin remain elevated. By immunostaining, the distributions of several glial proteins, including beta4 integrin, the voltage-gated potassium channel Kv1.1, and E-cadherin, are altered. Schwann cells from PMP22-deficient mice are able to produce limited amounts of myelin in DRG explant cultures, yet the internodal segments are dramatically fewer and shorter. The comparison of PMP22-deficient mice with other PMP22 mutant models reveals that the decrease in beta4 integrin is specific to an absence of PMP22. Furthermore, whereas lysosome-associated membrane protein 1 and ubiquitin are notably up-regulated in nerves of PMP22-deficient mice, heat shock protein 70 levels remain constant or decrease compared with wild-type or PMP22 mutant samples. Together these results support a role for PMP22 in the early events of peripheral nerve myelination. Additionally, although myelin abnormalities are a commonality among PMP22 neuropathic models, the underlying subcellular mechanisms are distinct and depend on the specific genetic abnormality.     

Axoglial interactions in myelin plasticity: Evaluating the relationship between neuronal activity and oligodendrocyte dynamics

Myelin is a critical component of the vertebrate nervous system, both increasing the conduction velocity of myelinated axons and allowing for metabolic coupling between the myelinating cells and axons. An increasing number of studies demonstrate that myelination is not simply a developmentally hardwired program, but rather that new myelinating oligodendrocytes can be generated throughout life. The generation of these oligodendrocytes and the formation of myelin are influenced both during development and adulthood by experience and levels of neuronal activity. This led to the concept of adaptive myelination, where ongoing activity-dependent changes to myelin represent a form of neural plasticity, refining neuronal functioning, and circuitry. Although human neuroimaging experiments support the concept of dynamic changes within specific white matter tracts relevant to individual tasks, animal studies have only just begun to probe the extent to which neuronal activity may alter myelination at the level of individual circuits and axons. Uncovering the role of adaptive myelination requires a detailed understanding of the localized interactions that occur between active axons and myelinating cells. In this review, we focus on recent animal studies that have begun to investigate the interactions between active axons and myelinating cells and review the evidence for—and against—the ability of neuronal activity to alter myelination at an axon-specific level.     

Changes in in vivo rates of protein synthesis on free and membrane-bound polysomes in rat brain during the development of physical dependence on ethanol and after the withdrawal of ethanol

Administration of ethanol and nutrients to rats thrice daily by gavage for 3 days produced a linear increase in physical dependence during the first 3 days and a 2% increase in body weight. Rates of protein synthesis on free and membrane-bound polysomes in whole brain and in 7 brain regions, comprising the entire brain, were measured in vivo under pool expansion conditions with [³H]leucine at intervals during the development and decay of ethanol dependence. During dependence development there was a progressive decrease in the rate of protein synthesis on free polysomes, but this change was not significant(P < 0.05) until the third day, and a decrease in the rate on membrane-bound polysomes after 3 days. The inhibition of protein synthesis is attributable to a decreased rate of polypeptide elongation. There was no change in brain weight, DNA content, ribosome content, ribosome distribution on mRNA size. There was, however, a decrease in leucine uptake after 3 days. In an attempt to distinguish between the acute effects of ethanol on regional rates of protein synthesis and those changes associated with dependence development, rates were measured 1.5 h after administering a 5 g/kg dose of ethanol to both control and dependent rats. Rates on free polysomes in the hippocampus-amygdala and thalamus-hypothalamus and on membrane-bound polysomes in the cerebellum and hipocampus-amygdala of dependent rats were reduced; however, there was a general reduction in the rates in control rats that may have obscured reductions in other regions from dependent rats. During early withdrawal, 12 h after the last dose of ethanol, there was an increase in the rate on free polysomes in the pons-medulla and striatum-septum and on membrane-bound polysomes in the hippocampus-amygdala, and a decrease in the rate on free polysomes in the cortex and thalamus-hypothalamus and on membrane-bound polysomes in the cortex. After 24 h, there was an increase in the rate on free polysomes in all regions (cerebellum, cortex, mesencephalon, striatum-septum and thalamus-hypothalamus) except the hippocampus-amygdala and pons-medulla and an increase in the rate on membrane-bound polysomes in all regions (cortex, hippocampus-amygdala, mesencephalon, pons-medulla and striatum-septum) except the cerebellum and thalamus-hypothalamus. The possible relationship of these changes to the homeostat hypothesis of ethanol dependence is discussed.