Differentially sensitive neuronal subpopulations in the central nervous system and the formation of hindbrain heterotopias in ethanol‐exposed zebrafish

Abstract

Background A cardinal feature of prenatal ethanol exposure is CNS damage, resulting in a continuum of neurological and behavioral impairments that are described by the term Fetal Alcohol Spectrum Disorders (FASD). FASDs are variable and depend on several factors, including the amount, timing and duration of prenatal ethanol exposure. To enhance interventions for CNS dysfunction, it is necessary to identify ethanol-sensitive neuronal populations and expand the understanding of factors that modify ethanol teratogenesis. Methods: To investigate the susceptibility of different neuronal subtypes, we exposed transgenic zebrafish (Danio rerio) to several ethanol concentrations (0.25%, 0.5%, 1.0%, 1.5% or 2.0%), at different hours post fertilization (hpf) (0 hpf, 6 hpf or 24 hpf), for various durations (0–24hpf, 0–48hpf, 4–24hpf, 6–24hpf, 6–48hpf or 24–48hpf). Following exposure, embryo survival rates were determined, and CNS neurogenesis, differentiation and patterning was assessed. Results: Embryo survival rates decrease as ethanol concentrations increase and drastically decline when exposed from 0–24hpf compared to 4–24hpf. Abnormal tangential migration of facial motor neurons is observed in isl1:gfp embryos exposed to ethanol concentrations as low as 0.25%, and the formation of IVth ventricle heterotopias are revealed by embryos exposed to ≥1.0% ethanol. Whereas, expression of olig2:dsred and ptf1a:gfp in the cerebellum and spinal cord are largely unaffected. While levels of etv4 mRNA are overtly resistant to ethanol, we observe significant reductions in ptch2 mRNA levels. Conclusions: These data show differentially sensitive CNS neuron subpopulations with susceptibility to low levels of ethanol. In addition, these data reveal the formation of ethanol-induced hindbrain heterotopias.

Embryonic Ethanol Exposure Affects the Early Development, Migration, and Location of Hypocretin/Orexin Neurons in Zebrafish

“…Whereas embryonic EtOH exposure is found to reduce whole‐brain levels of dopamine and serotonin in zebrafish (Buske and Gerlai, ), as well as reduce the number of cells expressing the pan‐neuronal marker elavl3:gfp (Buckley et al., ) and HuC + neurons in spinal cord (Joya et al., ), effects similarly observed in children with FASD (Riikonen et al., ) and rodents (Gil‐Mohapel et al., ; Livy et al., ), our results here in the AH reveal a very different effect, with 0.5% EtOH exposure found to increase the number of hcrt neurons, not only at 24 to 28 hpf but also at 30 hpf, 3 dpf, and 6 dpf. This stimulatory effect of low‐dose EtOH on the neurogenesis of orexigenic neuropeptides is consistent with our prior reports in both zebrafish (Sterling et al., ) and rodents (Chang et al., , ) and with another study showing EtOH consumption in rats to increase preproorexin mRNA in the lateral hypothalamus (Lawrence et al., ).…”

Maternal ethanol consumption before paternal fertilization: Stimulation of hypocretin neurogenesis and ethanol intake in zebrafish offspring

“…Studies with postconception ethanol generally reveal an inhibitory effect on neuronal development. This is shown in zebrafish by reduced number of HuC + neurons in spinal cord (Joya et al, 2014) and of cells expressing the pan-neuronal marker elavl3:gfp (Buckley et al, 2019) and reduced expression of the neural progenitor marker sox2 as well as HuC + and mature glial marker myelin-associated glycoprotein (Yin et al, 2014), results consistent with reports in children with fetal alcohol spectrum disorders (Riikonen et al, 2005) and rodents (Livy et al, 2003;Gil-Mohapel et al, 2010). Our observation here of preconception maternal ethanol consumption, showing no change in total number of differentiated neurons in HuC:GFP offspring along with an increase in hcrt neurogenesis, suggests that ethanol exposure before conception may affect specific, but not all, neuronal subpopulations during early development.…”

“…Based on previous studies showing postconception ethanol exposure to stimulate the neurogenesis of hcrt neurons (Chang et al, 2012;Sterling et al, 2016) while sometimes reducing overall neurogenesis (Gil-Mohapel et al, 2010;Buckley et al, 2019), we examined using live imaging in 28 hpf offspring of adult female zebrafish whether maternal consumption for 14 days of 10% ethanol-gelatin before conception alters the total number of differentiated neurons in HuC:GFP transgenic offspring and the number of hcrt neurons in transgenic hcrt:EGFP offspring. Compared to 545 ± 7.3 HuC + neurons detected in control animals at 28 hpf, we found that the offspring from the 10% ethanol-gelatin group had a similar number, an average of 542 ± 13.4 HuC + neurons, showing no significant difference (t( 18 ) = 0.2, ns) (Fig.…”

Zebrafish models of fetal alcohol spectrum disorders

“…While a 0.23 BAC dose is high, it is still physiologically relevant, since humans are capable of achieving much higher alcohol levels (Canfield, Forster, Cheong, & Cowan, 2019; A. W. Jones, 2008; Whaley, Young, & Gaynor, 2019). Work in zebrafish and other fish species has also shown media doses lower than 1% v/v impact embryonic development (Buckley, Sidik, Kar, & Eberhart, 2019; Dlugos, Brown, & Rabin, 2011; Dlugos & Rabin, 2003; Flentke et al, 2014; Gerlai, Ahmad, & Prajapati, 2008; Li et al, 2007; E. Loucks & Carvan, 2004; Oxendine et al, 2006; Reimers et al, 2004; Wang et al, 2006). Taken together, the data suggest that zebrafish researchers can generate target ethanol tissue concentrations that replicate human ethanol levels.…”

“…For example, ethanol exposure from gastrulation to pharyngeal stages (6–24 hr post fertilization [hpf]) disrupts formation of the mid‐hindbrain boundary (C. Zhang et al, 2013; Zhang, Anderson, & Cole, 2015) and reduces the number of elavl3‐positive neural progenitors (Joya, Garcia‐Algar, Vall, & Pujades, 2014). Using transgenic lines that label unique CNS neurons, Buckley et al (2019) found migration of facial branchial motor neurons to be ethanol sensitive. Additionally, the branching of secondary motor neurons increased and the diameter of Mautner axons decreased due to ethanol exposure (Shan, Boutin, Ferdous, & Ali, 2015).…”