Ethanol and Cannabinoids Regulate Zebrafish GABAergic Neuron Development and Behavior in a Sonic Hedgehog and Fibroblast Growth Factor-Dependent Mechanism

Abstract

Background: Ethanol (EtOH) has diverse effects on nervous system development, which includes development and survival of GABAergic neurons in a sonic hedgehog (Shh) and fibroblast growth factor (Fgf)-dependent mechanism. Cannabinoids also function as inhibitors of Shh signaling, raising the possibility that EtOH and cannabinoids may interact to broadly disrupt neuronal function during brain development.

Methods: Zebrafish embryos were exposed to a range of EtOH and/or cannabinoid receptor 1 (CB1R) agonist concentrations at specific developmental stages, in the absence or presence of morpholino oligonucleotides that disrupt shh expression. In situ hybridization was employed to analyze glutamic acid decarboxylase (gad1) gene expression as a marker of GABAergic neuron differentiation, and zebrafish behavior was analyzed using the novel tank diving test as a measure of risk-taking behavior.

Results: Combined acute subthreshold EtOH and CB1R agonist exposure results in a marked reduction in gad1 mRNA expression in zebrafish forebrain. Consistent with the EtOH and cannabinoid effects on Shh signaling, fgf8 mRNA overexpression rescues the EtOH- and cannabinoid-induced decrease in gad1 gene expression and also prevents the changes in behavior induced by EtOH and cannabinoids.

Conclusions: These studies provide evidence that forebrain GABAergic neuron development and zebrafish risk-taking behavior are sensitive to both EtOH and cannabinoid exposure in a Shh- and Fgf-dependent mechanism, and provide additional evidence that a signaling pathway involving Shh and Fgf crosstalk is a critical target of EtOH and cannabinoids in FASD.

Keywords: Cannabinoid; FASD; Fgf; Shh; gad1.

Figure 1:

Timeline for MO/ mRNA microinjection and ethanol and/or cannabinoid treatments are indicated, as well as times of morphological analysis of embryos (MHB and eye size) and behavioral testing.

Figure 2:

Fgf8 mRNA rescued small eye phenotype induced by combined acute exposure to shh MO and ethanol. Embryos were pre-injected with shh MO alone or with fgf8 mRNA and exposed to ethanol from 8–10 hpf and 24–27 hpf. Eye diameter was measured in 48 hpf embryos as described in Methods and representative photomicrographs are shown. A, wild-type embryos; B, embryos injected with shh MO alone at one- to two-cell stage; C, embryos injected with fgf8 mRNA alone at one- to two-cell stage; D, embryos exposed to 0.5% ethanol alone from 8–10 hpf; E, embryos exposed to 1% ethanol alone from 8–10 hpf; F, embryos exposed to 1 mg/L ACEA alone from 8–10 hpf; G, embryos exposed to 3 mg/L ACEA alone from 8–10 hpf; H, embryos injected with shh MO at one- to two-cell stage and exposed to 0.5% ethanol from 8–10 hpf; I, embryos injected with shh MO at one- to two-cell stage and exposed to 1% ethanol from 8–10 hpf; J, embryos co-injected with shh MO and fgf8 mRNA at one- to two-cell stage and exposed to 1% ethanol from 8–10 hpf; K, embryos exposed to 1 mg/L ACEA and 0.5% ethanol from 8–10 hpf; L, embryos exposed to 3 mg/L ACEA and 1% ethanol from 8–10 hpf; M, embryos exposed to both 3 mg/L ACEA and 1% ethanol from 8–10 hpf, and injected with fgf8 mRNA at the one- to two-cell stage; N, embryos exposed to 0.5% ethanol alone from 24–27 hpf; O, embryos exposed to 1% ethanol alone from 24–27 hpf; P, embryos exposed to 1 mg/L ACEA alone from 24–27 hpf; Q, embryos exposed to 3 mg/L ACEA alone from 24–27 hpf; R, embryos injected with shh MO at one- to two-cell stage and exposed to 0.5% ethanol from 24–27 hpf; S, embryos injected with shh MO at one- to two-cell stage and exposed to 1% ethanol from 24–27 hpf; T, embryos co-injected with shh MO and fgf8 mRNA at one- to two-cell stage and exposed to 1% ethanol from 24–27 hpf; U, embryos exposed to 1 mg/L ACEA and 0.5% ethanol from 24–27 hpf; V, embryos exposed to 3 mg/L ACEA and 1% ethanol from 24–27 hpf; W, embryos exposed to both 3 mg/L ACEA and 1% ethanol from 24–27 hpf, and injected with fgf8 mRNA at the one- to two-cell stage. Microphthalmia was observed only in embryos exposed to ethanol after shh MO injection (I and S) or exposed to both 3 mg/L ACEA and 1% ethanol (L and S). The small eye phenotype was rescued by fgf8 mRNA overexpression (J, M, T and W). The calibration bar is 50 μm. N≥ 30/group.

Figure 3:

Effects of ethanol exposure and shh MO treatments on eye diameter. The Mean eye diameter for each treatment group was quantified, with only combined ethanol and shh MO treatment resulting in a significant difference in eye diameter from other treatments, and fgf8 mRNA overexpression rescuing the effects of ethanol and shh MO treatments on eye diameter. A, 8–10 hpf ethanol exposure; B, 24–27 hpf ethanol exposure. ** significantly different from Control Mean, P< 0.001.

Figure 4:

Effects of ethanol and ACEA exposure on eye diameter. The Mean eye diameter for each treatment group was quantified, with only combined 3 mg/L ACEA + 1% ethanol treatment resulting in a significant difference in eye diameter from other treatments, and fgf8 mRNA overexpression rescuing the effects of combined ethanol and ACEA treatment on eye diameter. A, 8–10 hpf ethanol exposure; B, 24–27 hpf ethanol exposure. ** significantly different from Control Mean, P< 0.001.

Figure 5:

Gad1 gene expression is reduced in 24 hpf embryos following acute 1% ethanol and 3 mg/L ACEA co-exposure from 8–10 hpf and is rescued by fgf8 mRNA. Embryos were analyzed at 24 hpf for gad1 gene expression by whole-mount in situ hybridization. A, wild-type embryos; B, 0.5% ethanol alone; C, 1 mg/L ACEA alone; D, embryos injected with fgf8 mRNA alone at one- to two-cell stage; E, embryos exposed to 1% ethanol alone; F, embryos exposed to 3mg/L ACEA alone; G, embryos co-exposed to 0.5% ethanol and 3 mg/L ACEA; H, embryos co-exposed to 3 mg/L ACEA and 1% ethanol; I, embryos injected with fgf8 mRNA at one- to two-cell stage and co-exposed to 3 mg/L ACEA and 1% ethanol. Note: Decreased gad1 gene expression was only observed with combined 1% ethanol and 3 mg/L ACEA treatment, and fgf8 mRNA overexpression was able to reverse this reduced gene expression (F). N≥ 30/group.

Figure 6:

Quantitation of gad1 mRNA expression by quantitative RT-PCR. Gad1 mRNA levels were quantified by qRT-PCR and normalized to an internal ß-actin mRNA control. * significantly different from control expression, P< 0.05.

Figure 7:

Behavioral analysis of zebrafish embryos injected with shh MO and exposed to ethanol during late gastrulation to early neurulation (8–10 hpf). Embryos were assessed for novel tank diving response at 60–75 days. As controls, embryos were either untreated (Control), exposed to ethanol alone or injected with shh MO or fgf8 mRNA alone. Altered tank diving response, indicative of increased risk-taking behavior, is only observed for fish exposed to both shh MO and ethanol. Overexpression of fgf8 mRNA was able to attenuate the risk-taking behavior. Each bar represents the mean distance from the tank floor for one minute (min 1–5). Error bars represent SEM. (**significantly different from control (post-hoc) across all time points analyzed, P<0.001). N≥ 15 per group.

Figure 8:

Behavioral analysis of zebrafish embryos injected with shh MO and exposed to ethanol during late neurulation (24–27 hpf). Embryos were assessed for novel tank diving response at 60–75 days. As controls, embryos were either untreated (Control), injected with shh MO or fgf8 mRNA alone. Altered tank diving response is only observed for fish exposed to both shh MO and ethanol. Overexpression of fgf8 mRNA was able to attenuate the risk-taking behavior. Each bar represents the mean distance from the tank floor for one minute (min 1–5). Error bars represent SEM. (**significantly different from control (post-hoc) across all time points analyzed, P<0.001). N≥ 15 per group.

Figure 9:

Subthreshold ethanol and ACEA co-exposure during late gastrulation to early neurulation (8–10 hpf) alters risk-taking behavior in zebrafish embryos and is rescued by fgf8 mRNA overexpression. Embryos were exposed to ethanol and/or ACEA from 8–10 hpf and then assessed for novel tank diving response at 60–75 days. As controls, embryos were either untreated (Control) or exposed to fgf8 mRNA alone. Altered tank diving response, indicative of increased risk-taking behavior, is only observed for fish exposed to both ethanol and ACEA. Pre-injection of fgf8 mRNA was able to attenuate the risk-taking behavior. Each bar represents the mean distance from the tank floor for one minute (min 1–5). Error bars represent SEM. (**significantly different from control (post-hoc) across all time points analyzed, P<0.001). N≥ 15 per group.

Figure 10:

Subthreshold ethanol and ACEA co-exposure during late neurulation (24–27 hpf) alters risk-taking behavior in zebrafish embryos and is rescued by fgf8 mRNA overexpression. Embryos were exposed to ethanol and/or ACEA from 24–27 hpf and then assessed for novel tank diving response at 60–75 days. As controls, embryos were either untreated (Control) or exposed to fgf8 mRNA alone. Altered tank diving response, indicative of increased risk-taking behavior, is only observed for fish exposed to both ethanol and ACEA. Pre-injection of fgf8 mRNA was able to attenuate the risk-taking behavior. Each bar represents the mean distance from the tank floor for one minute (min 1–5). Error bars represent SEM. (**significantly different from control (post-hoc) across all time points analyzed, P<0.001). N≥ 15 per group.