Ketamine induces motor neuron toxicity and alters neurogenic and proneural gene expression in zebrafish

Abstract

Ketamine, a noncompetitive antagonist of N-methyl-d-aspartate-type glutamate receptors, is a pediatric anesthetic that has been shown to be neurotoxic in rodents and nonhuman primates when administered during the brain growth spurt. Recently, the zebrafish has become an attractive model for toxicity assays, in part because the predictive capability of the zebrafish model, with respect to chemical effects, compares well with that from mammalian models. In the transgenic (hb9:GFP) embryos used in this study, green fluorescent protein (GFP) is expressed in the motor neurons, facilitating the visualization and analysis of motor neuron development in vivo. In order to determine whether ketamine induces motor neuron toxicity in zebrafish, embryos of these transgenic fish were treated with different concentrations of ketamine (0.5 and 2.0 mm). For ketamine exposures lasting up to 20 h, larvae showed no gross morphological abnormalities. Analysis of GFP-expressing motor neurons in the live embryos, however, revealed that 2.0 mm ketamine adversely affected motor neuron axon length and decreased cranial and motor neuron populations. Quantitative reverse transcriptase-polymerase chain reaction analysis demonstrated that ketamine down-regulated the motor neuron-inducing zinc finger transcription factor Gli2b and the proneural gene NeuroD even at 0.5 mm concentration, while up-regulating the expression of the proneural gene Neurogenin1 (Ngn1). Expression of the neurogenic gene, Notch1a, was suppressed, indicating that neuronal precursor generation from uncommitted cells was favored. These results suggest that ketamine is neurotoxic to motor neurons in zebrafish and possibly affects the differentiating/differentiatedneurons rather than neuronal progenitors. Published 2011. This article is a US Government work and is in the public domain in the USA.

Figure 1

Effect of ketamine on zebrafish embryos. Ketamine does not have a drastic effect on zebrafish morphology but adversely affects the central nervous system as indicated by differences in relative GFP fluorescence. In the hb9:GFP transgenic fish motor neurons express hb9 promoter-driven GFP. Embryos at 28 hpf were treated with ketamine. After 20 h of treatment (48 hpf actual age), images of the live embryos were acquired for assessment of GFP fluorescence. In this experiment, MS-222 was not used (a routine procedure) to immobilize the untreated control embryos for photography in order to avoid any interference with the effect produced by ketamine. The experiment was repeated three times with n = 30 (replicates of 10 each) for each group in each experiment. Lateral views of the embryos are shown with dorsal side up. Embryos in different experimental groups are (A) control, (B) 0.5 mM ketamine-treated, and (C) 2.0 mM ketamine-treated. Scale bar = 280 μM.

Figure 2

Adverse effects of ketamine on cranial and spinal motor neurons. Hb9:GFP transgenic fish embryos (28 hpf) were treated with 2.0 mM ketamine. After 20 h of treatment (48 hpf actual age), images of the live embryos (lateral views with dorsal side up and anterior side to the left) were acquired for assessment of GFP fluorescence. GFP expressing motor neurons are shown in control (A) and ketamine-treated (B) brains, and control (C) and ketamine-treated (D) spinal cords. The arrows indicate the eyes, YS indicates yolk sac, and YE indicates yolk extension. Since the brain has a high density of GFP-positive motor neurons, the difference in motor neuron numbers could not be quantitatively determined. However, overall GFP fluorescence in ketamine-treated embryos was reduced compared with the untreated controls. Spinal motor neurons, however, could be visualized individually. Scale bar = 120 μM. Axon lengths from specific areas in the spinal cord region were measured using a micrometer in the microscope and the mean difference (%) between the control and ketamine-treated embryos from three different replications is presented (E). The value for the control does not have an error bar (SEM) because the spinal motor axon lengths were normalized relative to the lengths of the control. Student’s t-test was performed to determine statistical significance between observed differences and significance (*) was set at P < 0.05 (D).

Figure 3

Effect of ketamine on spinal motor neuron numbers. Embryos at 28 hpf were treated with 0.5 and 2.0 mM ketamine for 20 h (static exposure). Higher magnification of the trunk region showing GFP positive neurons in control (A), 0.5 mM ketamine treated- (B) and 2.0 mM ketamine-treated (C) embryos (48 hpf actual age). GFP-positive motor neurons in specific hemi-segments were counted, and Student’s t-test was performed to determine statistical significance between observed differences with significance (*) set at P < 0.05 (D). In each of three replications, spinal motor neurons were counted in 10 embryos each from the control and ketamine-treated groups. Scale bar = 30 μM.

Figure 4

Effect of ketamine on expression of specific proneural and neurogenic genes. Ketamine induces changes in gene expression in zebrafish embryos. Expression of the proneural gene Notch1a, and the neurogenic genes Ngn1, NeuroD, Gli2a and Gli2b at the mRNA level was analyzed. Embryos at 28 hpf were treated with 0.5 and 2.0 mM ketamine. After 20 h of treatment (48 hpf actual age), total RNA was isolated from control (untreated) and the two ketamine-treated groups. Following first-strand cDNA synthesis from the RNA, qPCR was performed. The 2−ΔΔC_t method was used to determine the relative gene expression. The GAPDH gene was the internal control for all qPCR experiments. The mean C_t values for GAPDH expression were 21.47 ± 0.11 (control), 21.28 ± 0.07 (0.5 mM ketamine) and 21.02 ± 0.073 (2 mM ketamine). Data from biological replicates were averaged and are shown as normalized gene expression ± SEM. For all pairwise multiple comparison procedures the Holm–Sidak method was used for data analysis with overall significance level set at P < 0.05. Lower-level ANOVAs were performed to determine further differences (between different ketamine doses).

Figure 5

Schematic representation of pathways potentially affected by ketamine. Ketamine has toxic effects on motor neurons in the zebrafish embryos. Down-regulation of the transmembrane receptor gene, Notch1a, could negatively affect ligand-dependent Notch signaling and inhibition of Notch signaling in the proneural domain is known to up-regulate the neurogenic gene, Ngn1, in the neuronal progenitors. Ngn1 is a direct inducer of NeuroD that is critical for neuronal differentiation and NeuroD is expressed in differentiated neurons. At the same time, in differentiated neurons, sustained inhibition of Notch signaling can adversely affect neuron survival. Ketamine-induced down-regulation of Gli2b, a mediator of Hedgehog signaling, can disrupt Hedgehog signaling, thereby adversely affecting motor neuron specification,differentiation and survival. Down-regulation of NeuroD expression in spite of an up-regulation of its inducer, Ngn1, could be the consequence of fewer surviving differentiated neurons.