Feng C. Zhou

Alcohol Teratogenesis: Mechanisms of Damage and Strategies for Intervention:

There are multiple mechanisms by which alcohol can damage the developing brain, but the type of damage induced will depend on the amount and developmental timing of exposure, along with other maternal and genetic factors. This article reviews current perspectives on how ethanol can produce neuroteratogenic effects by its interactions with molecular regulators of brain development. The current evidence suggests that alcohol produces many of its damaging effects by exerting specific actions on molecules that regulate key developmental processes (e.g., L1 cell adhesion molecule, alcohol dehydrogenase, catalase), interfering with the early development of midline serotonergic neurons and disrupting their regulatory-signaling function for other target brain structures, interfering with trophic factors that regulate neurogenesis and cell survival, or inducing excessive cell death via oxidative stress or activation of caspase-3 proteases. The current understanding of pathogenesis mechanisms suggests several strategic approaches to develop rational molecular prevention. However, the development of behavioral and biologic treatments for alcohol-affected children is crucial because it is unlikely that effective delivery of preventative interventions can realistically be achieved in ways to prevent prenatal damage in at-risk pregnancies. Toward that end, behavioral training that promotes experience-dependent neuroplasticity has been effective in a rat model of cerebellar damage induced by alcohol exposure during the period of brain development that is comparable to that of the human third trimester.

Serotonin transporters are located on the axons beyond the synaptic junctions : Anatomical and functional evidence

The serotonin (5-HT) transporter (5-HTT) is known to play a role in depression and many 5-HT related diseases, and is the target site for drugs of abuse, such as cocaine, MDMA, and methamphetamine. The major role of the 5-HTT has long been considered to be to inactivate serotonin transmission through the elimination of serotonin at release sites. However, immunocytochemistry using an antibody against the N-terminal of the 5-HTT at the light microscopic (LM) level indicates that the 5-HTT is associated not only with 5-HT varicosities but also with axons. Electron microscopy (EM) reveals that the majority of the 5-HTTs exist on the axolemma outside the synaptic junctions. In studying whether axonal 5-HTTs are involved in the uptake of 5-HT, we found with autoradiography that [3H]citalopram bound to all major 5-HT fibers, not only in the terminal regions, but also in 5-HT axonal bundles such as the cingulum bundle and medial forebrain bundle. Furthermore, voltammetry recordings indicated that serotonin axonal bundles were actively engaged in high affinity serotonin uptake. The evidence indicates that 5-HTTs on 5-HT axons away from the synapse are likely to be functional in a manner similar to the terminal 5-HTT for serotonin uptake. It also suggests that the role of the 5-HTT may not only be for the termination of synaptic transmission, but also for the regulation of 5-HT through extrasynaptic (volume) transmission. Our findings may also impact the understanding of the sites of action of selective serotonin reuptake inhibitors and drug entry into serotonin neurons via the numerous axonal sites.

Immunohistochemical detection of A1 adenosine receptors in rat brain with emphasis on localization in the hippocampal formation, cerebral cortex, cerebellum, and basal ganglia

Polyclonal antisera were generated against two identical regions of rat and human A1 adenosine receptors using synthetic multiple-antigenic-peptides as immunogens. Western blotting showed that the antisera recognized a single protein in brain of the expected size for A1 receptors. Immunohistochemistry of CHO cells transfected with the rat or human A1 adenosine receptor cDNAs showed robust labeling of the cell surface. In contrast, labeling was not apparent over non-transfected CHO cells, nor over CHO cells expressing A2a receptors. The pattern of immunoreactivity in rat brain was similar to that expected for A1 adenosine receptors. In contrast to receptor autoradiography or in situ hybridization methods, immunohistochemistry allowed identification of individually labeled cells and processes. Heavy labeling was apparent in many brain regions. In the hippocampal formation, strong labeling was present on granule cell bodies and dendrites, mossy fibers, and pyramidal neurons. In cerebellum, basket cells were the most heavily labeled cell type. Less intense staining was present over granule cells. In cerebral cortex, pyramidal cells were the most heavily labeled cell type, and some interneurons were also labeled. In the basal ganglia, 43% of neurons in the globus pallidus were labeled. In the caudate-putamen region, 38% of neurons were labeled. Heavy labeling was present in most thalamic nuclei, and moderate to heavy labeling was seen in many brainstem nuclei. These data identify specific cellular sites of A1 receptor expression and support the concept of cellular specificity of A1 adenosine receptor action.

Alcohol exposure alters DNA methylation profiles in mouse embryos at early neurulation

Alcohol exposure during development can cause variable neurofacial deficit and growth retardation known as fetal alcohol spectrum disorders (FASD). The mechanism underlying FASD is not fully understood. However, alcohol, which is known to affect methyl donor metabolism, may induce aberrant epigenetic changes contributing to FASD. Using a tightly controlled whole-embryo culture, we investigated the effect of alcohol exposure (88mM) at early embryonic neurulation on genome-wide DNA methylation and gene expression in the C57BL/6 mouse. The DNA methylation landscape around promoter CpG islands at early mouse development was analyzed using MeDIP (methylated DNA immunoprecipitation) coupled with microarray (MeDIP-chip). At early neurulation, genes associated with high CpG promoters (HCP) had a lower ratio of methylation but a greater ratio of expression. Alcohol-induced alterations in DNA methylation were observed, particularly in genes on chromosomes 7, 10, and X; remarkably, a >10 fold increase in the number of genes with increased methylation on chromosomes 10 and X was observed in alcohol-exposed embryos with a neural tube defect phenotype compared to embryos without a neural tube defect. Significant changes in methylation were seen in imprinted genes, genes known to play roles in cell cycle, growth, apoptosis, cancer, and in a large number of genes associated with olfaction. Altered methylation was associated with significant (p

Serotonergic sprouting is induced by dopamine-lesion in substantia nigra of adult rat brain

We have previously extracted a serotonin (5-HT) neurotrophic supernatant from the 5,7-DHT lesioned hippocampus. The current study shows that a new 5-HT neurotrophic signal was monitored in the striatum and nigra after DA-denervation. Such a signal may be involved in the heterotypic sprouting. Dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), was injected directly into the substantia nigra of adult rats. Two months after surgery, immunocytochemical staining showed that tyrosine hydroxylase (TH)-positive cell bodies had mostly disappeared in the substantia nigra, and TH-positive terminals in the striatum were almost completely depleted. Meanwhile, the 5-HT fibers, which exist in the same areas with low density, sprouted in the nigra as well as in the striatum and became dense. Normally 5-HT fibers innervate the striatum sparsely and the globus pallidus densely with sharp delineation (in the control side), and become dense across both areas with no appreciable delineation (in the lesion side). The increase of 5-HT fibers was more prominent in the posterior than in the anterior striatum. A significant increase in 5-HT and 5-HIAA levels was also evident in the posterior striatum when the decrease in DA level exceeded 90% in the nigra and striatum. In addition, we found that induction of 5-HT sprouting requires a greater than 90% decrease of DA level. Current data support that 6-OHDA injection in the substantia nigra of adult rats triggered a trophic signal or removed an inhibition for the growth of 5-HT neurons which responded with sprouting in the nigra as well as in the striatum.

Immunostained serotonergic fibers are decreased in selected brain regions of alcohol-preferring rats

The levels of serotonin (5-HT) and 5-hydroxyindole-acetic acid (5-HIAA) are decreased in the hippocampus, nucleus accumbens, and cortex of selectively bred alcohol-preferring (P) rats, compared with the alcohol-nonpreferring (NP) rats. In this study, we have confirmed these findings by immunocytochemistry and quantitative image analysis which indicates that there is a reduction of 5-HT immunostained fibers in several brain areas of alcohol-naive P rats. Three major areas possibly related to alcohol drinking, hippocampus, accumbens, and cortex, were examined. Pathways to these areas were also examined. The 5-HT fiber bundles had the same pattern in P and NP rats. However, in the terminal regions of the ventral hippocampus, the amount of 5-HT fibers was reduced in P rats as compared with NP rats. The 5-HT fibers in the hilus and CA4 of the dentate gyrus were also significantly decreased in the P rats. No differences in fiber density were seen in the anterior nucleus accumbens, but a significant decrease was seen in the middle medial and posterior accumbens of P rats. In the cortical regions examined, decreases in 5-HT fibers were observed in the posterior cingulum and anterior frontal cortex, but not in the insular frontal cortex of P rats. These observations indicate that there are quantitative decreases in 5-HT innervations or that the 5-HT in some 5-HT fibers is reduced to a level undetectable by immunocytochemistry in the brains of P rats when compared with that of NP rats.

Prenatal alcohol exposure retards the migration and development of serotonin neurons in fetal C57BL mice.

Incomplete neural tube fusion (iNTF), induced by alcohol, in midline floor and roof plates was found in our recent study. In this study, serotonin (5-HT) neurons, known to be born entirely in the midline raphe at brainstem, were examined during their development with fetal alcohol exposure. Weight-matched C57BL mice pregnant dams were divided into three groups on E8: one received ethanol via a chocolate Sustacal liquid diet providing 20% ethanol-derived calories as the sole source of nutrients (ALC); the second received an isocaloric Sustacal liquid diet and was pair-fed to individual dams in the ethanol-fed group (PF); the third was fed ad lib rat chow (Chow). Fetal brains were obtained on E15 and were processed for immunostaining of 5-HT and its trophic factor, S100 beta. The ascending 5-HT neurons, in normal development, appear bilaterally near midline on E12, and by E15, as seen in chow and PF groups, migrate from the midline germinal zone laterally and dorsally to their final position with rich fibers. In contrast, in the E15 ALC group, many 5-HT-im neurons were found remaining in the midline germinal region or had migrated, but with under-differentiated, sparse fibers. There were 20--30% fewer 5-HT-im neurons in ALC as compared to PF and Chow. In addition, the number of S100 beta cells was less in ALC as compared with PF and Chow groups. No difference was found between PF and Chow in number of 5-HT-im or S100 beta-im cells. The 5-HT neurons found compromised in migration and differentiation may, in part, stem from failure of access to floor plate or midline tissue induction and the insufficient support by S100 beta. As 5-HT neurons have been implicated for signaling brain maturation, fewer 5-HT neurons may have lasting effects on the development of brain or, if persistent in the adult, profoundly affect adult brain function.

Mesolimbic Dopamine System in Alcohol-Preferring Rats

The mesolimbic dopamine system of the brain, in particular the nucleus accumbens (Acb), is long known to be involved in reward behavior. When compared with the alcohol-nonpreferring NP rats, the alcohol-preferring P rats exhibit lower dopamine (DA) levels in the Acb as well as in other forebrain areas (20). In the present study, the DA innervation density, as determined by tyrosine hydroxylase (TH) immunostaining, was found lower in the selective cingulum cortex and the shell of the Acb of the P compared with that of NP rats. These structures cluster in the medial aspect of the mesolimbic system. There were no differences in other major DA mesolimbic brain regions. The subpopulation of DA neurons in the ventral tegmental area (VTA) projecting to the Acb was found to be smaller in the P than in the NP rats, as shown by horseradish peroxidase tracing and TH immunocytochemistry double staining. However, the total number of the DA neurons in the VTA, the major mesolimbic DA center, was found to be similar in the P and NP rats. These results indicate a selective reduction of catecholaminergic innervation in the dopaminergic medial mesolimbic system in the P rats and suggest that the P and NP rat lines should be a useful model for the investigation of DA involvement in alcohol drinking as well as other reinforced behaviors.

Four patterns of laminin-immunoreactive structure in developing rat brain

Laminin has been known to promote neurite outgrowth in culture. We have recently demonstrated that laminin facilitates and guides fiber growth of grafted neurons in the brain. In this study, I report that 4 distinct patterns of laminin immunoreactivity--small and large punctiform laminin, sheath laminin, and somal laminin--were expressed in the brain, each having unique spatial and temporal distributions. They are extensively produced in the developing brain while nerve fibers and vessels are actively growing. Two patterns of laminin, small and large puncta, disappear when the brain matures; the other two, sheath laminin and somal laminin, reduce in intensity but persist through adult life. The small puncta, size less than 1 micron in diameter, frequently accumulated along acellular spaces where future fiber bundles will form, interneuronal spaces and surfaces of neurons where terminal fibers dwell, and around the microvessels where neomicrovessels form. The small puncta, which reportedly appear at the eight-cell stage, were observed in our study at stage E10. They increased in expression from E14 to 19 during the stage when many nerve fibers are navigating to their targets, and subsided in the early postnatal days (P6) when less fiber growth occurs. Large puncta, size about 2 microns in diameter, existed exclusively in the hippocampus. They appeared about E16, and ended P16. The sheath laminin is known to form wrappings on microvessels, the ependymal layer, the choroid plexus, and the surface of the brain. This pattern of laminin attaches to non-neuronal structures which associate with CNS fluid. It was detected at E10 (our youngest group), increased its distribution along with expansion of the associated structure, and persisted throughout the adult life. The somal laminin, associated mainly with the soma of neurons, was the latest appearing laminin pattern (not until E16). It also persisted through adult life in the brain. The somal laminin, which was highly associated with the appearance of neuronal groups in brain nuclei, appeared group by group along with neurons. In summary, laminin attaches with distinct groups of cells in the central nervous system with specific temporal association. This close temporal and spatial association may allow laminin to actively affect nerve growth during brain development. The detailed mapping of laminin distribution is in progress in our lab.

Serotonin and its transporter on proliferation of fetal heart cells

Besides neuronal transmission, serotonin (5‐HT) also acts as a trophic signal during the development of the central nervous and neural crest systems. In this study, we report that in addition to trophic effect, 5‐HT increases the proliferation of fetal heart cells. We showed for the first time that the cultured heart cells, express serotonin transporter (5‐HTT), which confirmed the previously observed accumulation of 5‐HT in developing heart. The influence of 5‐HT on developing heart cells is studied throughout the dosage. We found that 5‐HT concentration at physiological level, 4 μM, permits an optimal proliferation of heart cells as indicated by the number of 5‐bromo‐deoxyuridine immunoreactive (BrdU‐im) cells and myosin heavy chain immunoreactive cells (MF20‐im); fluctuation towards either concentrations reduce the proliferation. We hypothesized that 5‐HTT plays a role in the heart development. Our study indicated that the blockade of 5‐HT uptake by paroxetine decreased the number of BrdU‐im cells and MF20‐im cells. These data indicate a role of 5‐HT and 5‐HTT on heart development. Abnormal 5‐HT level or misuse of 5‐HT uptake blocker may alter the heart development.