Chronic prenatal ethanol exposure-induced decrease of guinea pig hippocampal CA1 pyramidal cell and cerebellar Purkinje cell density

The brain is a key target of ethanol teratogenicity, in which ethanol can produce neurodegeneration in selected areas, including the hippocampus and cerebellum. The research objective was to test the hypothesis that chronic prenatal ethanol exposure, via maternal ethanol administration, produces differential time course of decreased linear density of hippocampal CA1 pyramidal cells and cerebellar Purkinje cells. Timed pregnant guinea pigs received chronic oral administration of ethanol, isocaloric-sucrose/pair-feeding, or water throughout gestation (term, about gestational day (GD) 68), and the offspring were studied at GD 62 (near-term fetus), postnatal day (PD) 1 (neonate), PD 5, and PD 12 (early postnatal life). Ethanol treatment, compared with isocaloric-sucrose/pair-feeding and water treatments, decreased brain, hippocampal, and cerebellar weights at GD 62, PD 1, PD 5, and PD 12. Hippocampal CA1 pyramidal cell linear density and cerebellar Purkinje cell linear density were unaffected at GD 62. Ethanol treatment produced 25, 30, and 30% decreases in linear density of hippocampal CA1 pyramidal cells at PD 1, PD 5, and PD 12, respectively, and a 30% decrease in linear density of cerebellar Purkinje cells at PD 12 only. At PD 5, Purkinje cell profile linear density remained unaffected; however, ethanol treatment appeared to increase linear density of apoptotic Purkinje cell nuclei, as determined by a modified TUNEL method. The data demonstrate that chronic prenatal ethanol exposure produces apparent differential time course of decreased linear density of hippocampal CA1 pyramidal cells and cerebellar Purkinje cells in the developing guinea pig.     

A longitudinal study of the effects of prenatal ethanol exposure on neuronal acquisition and death in the principal sensory nucleus of the trigeminal nerve: interaction with changes induced by transection of the infraorbital nerve

The present study determines (1) whether ethanol-induced microencephaly results from reductions in neuronal acquisition (i.e., cell proliferation and neuronal migration) and/or increases in neuronal death and (2) whether ethanol exacerbates death by the same mode as that for naturally occurring or lesion-induced neuronal death. Pregnant rats were exposed to a diet containing 6.7% (v/v) ethanol or an isocaloric control diet during the last two weeks of gestation. At birth, the right infraorbital nerves of the pups were transected. The numbers of neurons in the principal sensory nucleus of the trigeminal nerve (PSN) on both sides of the pons were examined at various prenatal and early postnatal timepoints. The numbers of pyknotic and argyrophilic PSN cells were also counted. Ethanol delayed and reduced (19.9%) the prenatal acquisition of PSN neurons. The postnatal decline in neuronal number (indicative of neuronal death) was significantly increased (10.6%) by ethanol. Likewise, the numbers of pyknotic and silver-stained cells were significantly higher in ethanol-treated rats. Lesion of the infraorbital nerve induced significant transsynaptic neuronal death in the control rats. Ethanol increased the amount of death caused by the lesion; however, it altered neither the timing of the neuronal loss nor the incidence of pyknosis or silver-staining. Therefore, ethanol affects both neuronal acquisition and survival; the greater effect being on neuronal acquisition. The timing and morphology of dying cells indicate that regardless of the cause (natural processes, ethanol-induced, or lesion-induced), neurons die in the developing PSN by the same mode.     

Ethanol influences on the chick embryo spinal cord motor system: Analyses of motoneuron cell death,motility, and target trophic factor activity and in vitro analyses of neurotoxicity and trophic factor neuroprotection

A series of in vivo and in vitro experiments were conducted to determine the influence of prenatally administered ethanol on several aspects of the developing chick embryo spinal cord motor system. Specifically, we examined: (1) the effect of chronic ethanol administration during the natural cell death period on spinal cord motoneuron numbers; (2) the influence of ethanol on ongoing embryonic motility; (3) the effect of ethanol exposure on neurotrophic activity in motoneuron target tissue (limbbud); and (4) the responsiveness of cultured spinal cord neurons to ethanol, and the potential of target-derived neurotrophic factors to ameliorate ethanol neurotoxicity. These studies revealed the following: Chronic prenatal ethanol exposure reduces the number of motoneurons present in the lateral motor column after the cell death period [embryonic day 12 (E12)]. Ethanol tends to inhibit embryonic motility, particularly during the later stages viewed (E9-E11). Chronic ethanol exposure reduces the neurotrophic activity contained in target muscle tissue. Such diminished support could contribute to the observed motoneuron loss. Direct exposure of spinal cord neurons to ethanol decreases neuronal survival and process outgrowth in a dose-dependent manner, but the addition of target muscle extract to ethanol-containing cultures can ameliorate this ethanol neurotoxicity. These studies demonstrate ethanol toxicity in a population not previously viewed in this regard and suggest a mechanism that may be related to this cell loss (i.e., decreased neurotrophic support). © 1995 John Wiley & Sons, Inc.     

Ethanol-induced face-brain dysmorphology patterns are correlative and exposure-stage dependent

Prenatal ethanol exposure is the leading preventable cause of congenital mental disability. Whereas a diagnosis of fetal alcohol syndrome (FAS) requires identification of a specific pattern of craniofacial dysmorphology, most individuals with behavioral and neurological sequelae of heavy prenatal ethanol exposure do not exhibit these defining facial characteristics. Here, a novel integration of MRI and dense surface modeling-based shape analysis was applied to characterize concurrent face-brain phenotypes in C57Bl/6J fetuses exposed to ethanol on gestational day (GD)7 or GD8.5. The facial phenotype resulting from ethanol exposure depended upon stage of insult and was predictive of unique patterns of corresponding brain abnormalities. Ethanol exposure on GD7 produced a constellation of dysmorphic facial features characteristic of human FAS, including severe midfacial hypoplasia, shortening of the palpebral fissures, an elongated upper lip, and deficient philtrum. In contrast, ethanol exposure on GD8.5 caused mild midfacial hypoplasia and palpebral fissure shortening, a shortened upper lip, and a preserved philtrum. These distinct, stage-specific facial phenotypes were associated with unique volumetric and shape abnormalities of the septal region, pituitary, and olfactory bulbs. By demonstrating that early prenatal ethanol exposure can cause more than one temporally-specific pattern of defects, these findings illustrate the need for an expansion of current diagnostic criteria to better capture the full range of facial and brain dysmorphology in fetal alcohol spectrum disorders.     

NeuroD regulates neuronal migration

NeuroD is required for the survival of many subtypes of developing neurons in the vertebrate central nervous system. Because NeuroD-deficient neurons in the hippocampus, cerebellum, and inner ear die prematurely in the early stage of neurogenesis, the role of NeuroD during the later stages of neurogenesis of these cell subtypes is not well understood. In addition, the mechanism of NeuroD-deficient neuronal death has not been investigated. It was hypothesized that NeuroD-dependent neuronal death occurs through a Bax-dependent apoptotic pathway. Based on this hypothesis, this study attempted to rescue neuronal cell death by deleting the Bax gene in NeuroD null mice to investigate the role of NeuroD in surviving neurons. The NeuroD and Bax double null mice displayed a decrease in the number of apoptotic cells in the hippocampus and the cerebellum and the rescue of vestibulocochlear ganglion (VCG) neurons that failed to migrate and innervate. In addition, at E13.5, the NeuroD^−/−Bax^−/− VCG neurons failed to express TrkB and TrkC, which are known to be essential for the survival of those neurons. These data suggest that neuronal death in NeuroD null mice is mediated by Bax-dependent apoptosis and that NeuroD is required for the migration of VCG neurons. Finally, these data show that TrkB and TrkC expression in E13.5 VCG neurons requires NeuroD and that TrkB and TrkC expression may be necessary for the normal migration and innervations of those neurons.     

Coenzyme Q10 Protects Hair Cells against Aminoglycoside

It is well known that the production of free radicals is associated with sensory cell death induced by an aminoglycoside. Many researchers have reported that antioxidant reagents protect sensory cells in the inner ear, and coenzyme Q10 (CoQ10) is an antioxidant that is consumed as a health food in many countries. The purpose of this study was to investigate the role of CoQ10 in mammalian vestibular hair cell death induced by aminoglycoside. Cultured utricles of CBA/CaN mice were divided into three groups (control group, neomycin group, and neomycin + CoQ10 group). In the neomycin group, utricles were cultured with neomycin (1 mM) to induce hair cell death. In the neomycin + CoQ10 group, utricles were cultured with neomycin and water-soluble CoQ10 (30–0.3 µM). Twenty-four hours after exposure to neomycin, the cultured tissues were fixed, and vestibular hair cells were labeled using an anti-calmodulin antibody. Significantly more hair cells survived in the neomycin + CoQ10 group than in the neomycin group. These data indicate that CoQ10 protects sensory hair cells against neomycin-induced death in the mammalian vestibular epithelium; therefore, CoQ10 may be useful as a protective drug in the inner ear.     

The zinc finger transcription factor Gfi1, implicated in lymphomagenesis,is required for inner ear hair cell differentiation and survival

Gfi1 was first identified as causing interleukin 2-independent growth in T cells and lymphomagenesis in mice. Much work has shown that Gfi1 and Gfi1b, a second mouse homolog, play pivotal roles in blood cell lineage differentiation. However, neither Gfi1 nor Gfi1b has been implicated in nervous system development, even though their invertebrate homologues, senseless in Drosophila and pag-3 in C. elegans are expressed and required in the nervous system. We show that Gfi1 mRNA is expressed in many areas that give rise to neuronal cells during embryonic development in mouse, and that Gfi1 protein has a more restricted expression pattern. By E12.5 Gfi1 mRNA is expressed in both the CNS and PNS as well as in many sensory epithelia including the developing inner ear epithelia. At later developmental stages, Gfi1 expression in the ear is refined to the hair cells and neurons throughout the inner ear. Gfi1 protein is expressed in a more restricted pattern in specialized sensory cells of the PNS, including the eye, presumptive Merkel cells, the lung and hair cells of the inner ear. Gfi1 mutant mice display behavioral defects that are consistent with inner ear anomalies, as they are ataxic, circle, display head tilting behavior and do not respond to noise. They have a unique inner ear phenotype in that the vestibular and cochlear hair cells are differentially affected. Although Gfi1-deficient mice initially specify inner ear hair cells, these hair cells are disorganized in both the vestibule and cochlea. The outer hair cells of the cochlea are improperly innervated and express neuronal markers that are not normally expressed in these cells. Furthermore, Gfi1 mutant mice lose all cochlear hair cells just prior to and soon after birth through apoptosis. Finally, by five months of age there is also a dramatic reduction in the number of cochlear neurons. Hence, Gfi1 is expressed in the developing nervous system, is required for inner ear hair cell differentiation, and its loss causes programmed cell death.     

Proliferation and death of cultured fetal neocortical neurons: effects of ethanol on the dynamics of cell growth

Neuronal number in the mature CNS is determined by the balance of cell proliferation and death. The effects of ethanol on cell proliferation and death were examined in primary cultures of neocortical neurons derived from 16-day-old rat fetuses. The cells were treated with ethanol (0 or 400 mg/dl) and examined for (1) immunohistochemical identity, (2) cell cycle kinetics using a cumulative bromodeoxyuridine labeling technique, (3) viable cell number via a trypan blue assay, and (4) the incidence of cell death with terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and caspase 3 immunhistochemistry. After two days in culture, most (>85%) cells expressed a neuron-specific antigen(s) whether or not ethanol was added to the culture medium. Ethanol affected the proliferation of the cultured cells, e.g., the length of the cell cycle was greater in the ethanol-treated cells than in controls. The number of trypan blue-negative (viable) cells was profoundly decreased by ethanol exposure. This decrease was accompanied by increases in the frequencies of TUNEL- and caspase 3-positive cells and of cells exhibiting nuclear condensations. Thus, ethanol decreases the number of viable cells in vitro by slowing cell proliferation and increasing the incidence of cell death. The expression of the death indices in untreated cultures is most consistent with a single (apoptotic) pathway of cell death, rather than simultaneous apoptotic and necrotic modes of death. Furthermore, it appears that ethanol initiates an apoptotic death among cultured cortical neurons.     

Impaired development of mitochondria plays a role in the central nervous system defects of fetal alcohol syndrome

BACKGROUND: Alcohol consumption during pregnancy can induce a wide spectrum of adverse effects in offspring. Microcephaly and mental retardation are two major defects of central nervous system (CNS). Most mechanism studies of alcohol-related CNS defects have been focused on the morphologically abnormal tissues, and more attention has been paid to nuclear alteration as opposed to organelle development. METHODS: A mouse model of fetal alcohol syndrome (FAS) was used to investigate the effect of alcohol on fetal cerebral mitochondria development. Pregnant mice were given different doses of ethanol intragastrically from GD6 to GD15. Fetal cerebral mitochondria were isolated and analyzed on GD18. RESULTS: Excessive cell apoptosis was found in the cerebra of prenatal alcohol exposure fetuses. Proliferation and differentiation of fetal cerebral mitochondria were inhibited by alcohol. Affected mitochondrial volume constriction and adenosine triphosphate (ATP) accumulation, reduced activities of respiratory chain complex I and IV and ATP synthase were detected in the cerebral tissue without obvious malformed appearance. CONCLUSIONS: Impaired mitochondria development plays a role in the CNS defects induced by prenatal alcohol exposure.     

Development of the inner ear efferent system across vertebrate species

Inner ear efferent neurons are part of a descending centrifugal pathway from the hindbrain known across vertebrates as the octavolateralis efferent system. This centrifugal pathway terminates on either sensory hair cells or eighth nerve ganglion cells. Most studies of efferent development have used either avian or mammalian models. Recent studies suggest that prevailing notions of the development of efferent innervation need to be revised. In birds, efferents reside in a single, diffuse nucleus, but segregate according to vestibular or cochlear projections. In mammals, the auditory and vestibular efferents are completely separate. Cochlear efferents can be divided into at least two distinct, descending medial and lateral pathways. During development, inner ear efferents appear to be a specific motor neuron phenotype, but unlike motor neurons have contralateral projections, innervate sensory targets, and, at least in mammals, also express noncholinergic neurotransmitters. Contrary to prevailing views, newer data suggest that medial efferent neurons mature early, are mostly, if not exclusively, cholinergic, and project transiently to the inner hair cell region of the cochlea before making final synapses on outer hair cells. On the other hand, lateral efferent neurons mature later, are neurochemically heterogeneous, and project mostly, but not exclusively to the inner hair cell region. The early efferent innervation to the ear may serve an important role in the maturation of afferent responses. This review summarizes recent data on the neurogenesis, pathfinding, target selection, innervation, and onset of neurotransmitter expression in cholinergic efferent neurons.     

Stem cells for the replacement of inner ear neurons and hair cells

Stem cells in the nervous system have some capacity to restore damaged tissue. Proliferation of stem cells endows them with self-renewal ability and accounts for in vitro formation of neurospheres, clonally derived colonies of floating cells. However, damage to the nervous system is not readily repaired, suggesting that the stem cells do not provide an easily recruited source of cells for regeneration. The vestibular and auditory organs, despite their limited ability to replace damaged cells, appear to contain cells with stem cell properties. These inner ear stem cells, identified by neurosphere formation and by their expression of markers of inner ear progenitors, can differentiate to hair cells and neurons. Differentiated cells obtained from inner ear stem cells expressed sensory neuron markers and, after co-culture with the organ of Corti, grew processes that extended to hair cells. The neurons expressed synaptic vesicle markers at points of contact with hair cells. Exogenous stem cells have also been used for hair cell and neuron replacement. Embryonic stem cells are one potential source of both hair cells and sensory neurons. Neural progenitors made from embryonic stem cells, transplanted into the inner ear of gerbils that had been de-afferented by treatment with a toxin, differentiated into cells that expressed neuronal markers and grew processes both peripherally into the organ of Corti and centrally. The regrowth of these neurons suggests that it may be possible to replace auditory neurons that have degenerated with neurons that restore auditory function by regenerating connections to hair cells.     

Pathogenesis of methanol-induced craniofacial defects in C57BL/6J mice

BACKGROUND: Methanol administered to C57BL/6J mice during gastrulation causes severe craniofacial dysmorphology. We describe dysmorphogenesis, cell death, cell cycle assessment, and effects on development of cranial ganglia and nerves observed following administration of methanol to pregnant C57BL/6J mice on gestation day (GD) 7. METHODS: Mice were injected (i.p.) on GD 7 with 0, 2.3, 3.4, or 4.9 gm/kg methanol, split into two doses. In embryos of mice treated with 0 or 4.9 gm/kg methanol, we used histology and LysoTracker red staining on GD 8 0 hr through GD 8 18 hr to examine cell death and dysmorphogenesis, and we also evaluated cell-cycle distribution and proliferation using flow cytometry (FCM) and BrdU immunohistochemistry. On GD 10, we evaluated the effect of GD 7 exposure to 0, 2.3, 3.4, or 4.9 gm/kg methanol on cranial ganglia and nerve development using neurofilament immunohistochemistry. RESULTS: Methanol treatment on GD 7 resulted in reduced mesenchyme surrounding the fore- and midbrain, and in the first branchial arches, by GD 8 12 hr. There were disruptions in the forebrain neuroepithelium and optic pit. Neural crest cell emigration from the mid- and hindbrain region was reduced in methanol-exposed embryos. Methanol had no apparent effect on BrdU incorporation or cell-cycle distribution on GD 8. Cell death was observed in the hindbrain region along the path of neural crest migration and in the trigeminal ganglion on GD 8 18 hr. Development of the cranial ganglia and nerves was adversely affected by methanol. Development of ganglia V, VIII, and IX was decreased at all dosage levels; ganglion VII was reduced at 3.4 and 4.9 gm/kg, and ganglion X was reduced at 4.9 gm/kg. CONCLUSIONS: These results suggest that gastrulation-stage methanol exposure affects neural crest cells and the anterior mesoderm and neuroepithelium. Cell death was evident in areas of migrating neural crest cells, but only at time points after methanol was cleared from the embryo, suggesting an indirect effect on these cells. Birth Defects Research (Part A), 2004. Published 2004 Wiley-Liss, Inc.     

Mechanism of ethanol enhancement of apoptosis and caspase activation in serum-deprived PC12 cells

Neuronal death is one of the most prominent consequences of alcohol exposure during development. Ethanol-induced neuronal death appears to involve apoptosis. The objective of the present study was to characterize the effect of ethanol on neuronal cell viability and to determine the mechanism by which ethanol enhances apoptosis in neural cells. For these studies the rat pheochromocytoma (PC12) cells were used. PC12 cells were incubated for 24 h in the presence or absence of 100 mM ethanol. Apoptosis was induced by serum withdrawal. Ethanol in the presence of serum-containing media did not alter cell viability, while incubation of PC12 cells in serum-free media resulted in a significant increase in cell death that was further significantly increased by 35% in cells exposed to ethanol. The temporal response of the PC12 cells to serum withdrawal was studied over a period of 22 h. At least 18 h of ethanol exposure was necessary to observe a significant increase in death for cells incubated in serum-free media. An increase in the caspase-3 activity in PC12 cells deprived of serum was observed that was further increased by ethanol exposure. This increase of caspase-3 activity was correlated with an enhancement of caspase-9 activity. Ethanol exposure increased the amount of cytosolic cytochrome c in PC12 cells incubated in serum-free media but did not alter the level of cytochrome c in cells incubated in serum. Finally, a 26% increase was observed in the number of cells with depolarized mitochondria due to ethanol treatment. The present study implicates an increase in the mitochondrial outer membrane permeability as a potential mechanism of enhancement of apoptosis in serum-deprived PC12 cells by ethanol.     

Effect of maternal diabetes and ethanol interactions on embryo development in the mouse

The aim of this study was to determine the possible fetal effects of interaction between maternal diabetes and acute doses of alcohol. Pregnant TO mice were made diabetic by a single injection of streptozotocin (STZ) on gestation day (GD) 2. Single dose of 0.003 or 0.03 ml/g body weight of fresh ethanol (25% v/v of absolute alcohol in normal saline) was injected into groups of diabetic and nondiabetic animals on GD 7 or 8. One group of diabetic animals had a daily dose of 6-8 IU of insulin subcutaneously. Fetuses were collected on GD 18. There was a significant increase in the incidence of implantation failure in the diabetes plus ethanol groups and insulin control group. Ethanol injection on GD 7 accentuated diabetes-related embryonic resorption and intrauterine growth retardation (IUGR). This effect was less marked in the diabetic group treated with ethanol on GD 8. Diabetes alone produced a greater incidence of IUGR than ethanol alone. Midfacial hypoplasia and minor anomalies were found more frequently in the combination treatment groups. Holoprosencephaly and thymus hypoplasia observed in diabetic groups were found to be reduced in frequency in the diabetes plus ethanol groups, suggesting an antagonistic type of ethanol-diabetes interaction, stage-dependently. Since severely malformed embryos are known to be resorbed/killed in utero in mice, this reduction might reflect the magnitude of early death of severely malformed embryos. These data suggest that the interaction effects are possibly related to alterations in fundamental developmental processes of early embryos.     

Differential expression of LIM domain-only (LMO) genes in the developing mouse inner ear

The vertebrate inner ear, a complex sensory organ with vestibular and auditory functions, is derived from a single ectoderm structure called the otic placode. Currently, the molecular mechanisms governing the differentiation and specification of the otic epithelium are poorly understood. We present here a detailed expression study of LMO1-4 in the developing mouse inner ear using a combination of in situ hybridization and immunohistochemistry. LMO1 is specifically expressed in the vestibular and cochlear hair cells as well as the vestibular ganglia of the developing inner ear. LMO2 expression is detected in the periotic mesenchyme of the developing mouse cochlea from E12.5 to E14.5. The expression of LMO3 expression is first observed in the cochlea at E13.5 and becomes confined to the lesser epithelial ridge (LER) from E14.5 to E17.5. LMO3 is also expressed in some of the vestibular ganglion cells. LMO4 is initially expressed in the dorsolateral portion of the otic vesicle and its expression persists in the semicircular canals, macula, crista, and the spiral ganglia throughout embryogenesis. Thus, the regionalized expression patterns of LMO1-4 are closely associated with the morphogenesis of the inner ear.     

Clonal analysis of the relationships between mechanosensory cells and the neurons that innervate them in the chicken ear

In vertebrates, hair-cell-bearing mechanosensory organs and the neurons that innervate them share a common placodal origin. In the inner ear, the peripheral neurons for both auditory and vestibular systems emigrate from the otic placode as neuroblasts, and divide, differentiate and innervate only one of six to eight distinct sensory organs. How these neurons find their correct target is unknown, although one suggestion is that they synapse with clonally related cells. To test this idea for both the middle and inner ears of chicken embryos, lineage analysis was initiated at the time of neuroblast delamination by labeling progenitors with replication-defective retroviruses. The vast majority (89%) of clones were restricted to a single anatomical subdivision of the sensory periphery or its associated ganglia, indicating limited clonal dispersion. Among the remaining clones, we found evidence of a shared neurosensory lineage in the middle ear. Likewise, in the inner ear, neurons could be related to cells of the otic epithelium, although the latter cells were not widely distributed. Rather, they were restricted to a region in or near the utricular macula. None of the other seven sensory organs was related to the ganglion neurons, suggesting that a common lineage between neurons and their targets is not a general mechanism of establishing synaptic connections in the inner ear. This conclusion is further strengthened by finding a shared lineage between the vestibular and acoustic ganglia, revealing the presence of a common progenitor for the two functional classes of neurons.