Distinct effects of ketamine and acetyl L-carnitine on the dopamine system in zebrafish

Abstract

Ketamine, a noncompetitive N-methyl-D-aspartic acid (NMDA) receptor antagonist is commonly used as a pediatric anesthetic. We have previously shown that acetyl L-carnitine (ALCAR) prevents ketamine toxicity in zebrafish embryos. In mammals, ketamine is known to modulate the dopaminergic system. NMDA receptor antagonists are considered as promising anti-depressants, but the exact mechanism of their function is unclear. Here, we measured the levels of dopamine (DA) and its metabolites, 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in the zebrafish embryos exposed to ketamine in the presence and absence of 0.5 mM ALCAR. Ketamine, at lower doses (0.1-0.3 mM), did not produce significant changes in DA, DOPAC or HVA levels in 52 h post-fertilization embryos treated for 24 h. In these embryos, tyrosine hydroxylase (TH) mRNA expression remained unchanged. However, 2 mM ketamine (internal embryo exposure levels equivalent to human anesthetic plasma concentration) significantly reduced DA level and TH mRNA indicating that DA synthesis was adversely affected. In the presence or absence of 2 mM ketamine, ALCAR showed similar effects on DA level and TH mRNA, but increased DOPAC level compared to control. ALCAR reversed 2 mM ketamine-induced reduction in HVA levels. With ALCAR alone, the expression of genes encoding the DA metabolizing enzymes, MAO (monoamine oxidase) and catechol-O-methyltransferase (COMT), was not affected. However, ketamine altered MAO mRNA expression, except at the 0.1 mM dose. COMT transcripts were reduced in the 2 mM ketamine-treated group. These distinct effects of ketamine and ALCAR on the DA system may shed some light on the mechanism on how ketamine can work as an anti-depressant, especially at sub-anesthetic doses that do not affect DA metabolism and suppress MAO gene expression.

Keywords: Acetyl l-carnitine; Dopamine; Ketamine; MAO; Tyrosine hydroxylase; Zebrafish.

Fig. 1

Effects of low doses of ketamine on DA, DOPAC and HVA levels in zebrafish embryos. Embryos at 52 hpf were treated with 0.1–0.3 mM ketamine. Twenty four hours post exposure (76 hpf) embryos were processed for DA (A) as well as its metabolites, DOPAC (B) and HVA (C) measurements using HPLC/EC. Values are expressed in ng/µg protein in embryo extracts (means ± SDs). One-way ANOVA with post-hoc analyses were used to assess statistical significance in variation in DA, DOPAC and HVA levels between the experimental groups as well as with controls.

Fig. 2

Effects of ketamine at a dose relevant to the human anesthetic dose and ALCAR on DA, DOPAC and HVA levels in zebrafish embryos. Embryos at 52 hpf were treated with 2 mM ketamine, 2 mM ketamine plus 0.5 mM ALCAR or 0.5 mM ALCAR alone. Twenty four hours post exposure (76 hpf), embryos were processed for DA (A) as well as its metabolites, DOPAC (B) and HVA (C) measurements using HPLC/EC. Values are expressed in ng/µg protein in the embryo extracts as means ± SDs. One-way ANOVA with post-hoc analyses were used to assess statistical significance (*P = <0.05; **P = <0.001; ***P = <0.0001) of variations in levels of DA and its metabolites, DOPAC and HVA between the experimental groups as well as with controls.

Fig. 3

Effects of ketamine and ALCAR on DA metabolism in zebrafish embryos. Embryos at 52 hpf were treated with lower doses of ketamine (0.1–0.3 mM; upper panels) or 2 mM ketamine, 2 mM ketamine plus 0.5 mM ALCAR or 0.5 mM ALCAR alone (lower panels). Twenty hours post exposure (76 hpf) embryos were processed for DA as well as its metabolites, DOPAC and HVA measurements using HPLC/EC. The rate of DA metabolism was modeled using the metabolite/DA ratios. The ratios for the embryos treated with 0.1– 0.3mM ketamine are presented in the top panels, (A) DOPAC/DA and (C) HVA/DA. The ratios for the embryos treated with 2 mM ketamine, 2 mM ketamine plus 0.5 mM ALCAR or 0.5 mM ALCAR alone are presented in the lower panels, (B) DOPAC/DA and (D) HVA/DA. Values are expressed as means ± SDs. One-way ANOVA with post-hoc analyses were used to assess statistical significance (*P = <0.05; **P = <0.001; ***P= <0.0001) of the variation in values between the experimental groups as well as with the controls.

Fig. 4

Effects of ketamine and ALCAR on TH gene expression. Embryos at 52 hpf were treated with lower doses of ketamine (0.1–0.3 mM) (A) as well as 2 mM ketamine, 2 mM ketamine plus 0.5 mM ALCAR or 0.5 mM ALCAR (B) alone for 24 h. Total RNA was extracted from the embryos (76 hpf) and after first strand cDNA synthesis from the RNA, quantitative real-time polymerase chain reaction (qRT-PCR) was performed. The 2-ΔΔCT method was used to determine relative gene expression for TH. The GAPDH gene served as the internal control for all qRT-PCR experiments. Data were averaged and shown as normalized (fold-change over control) relative gene expression ± SDs. One-way ANOVA with comparison post-hocs were used to determine statistical significance with P = <0.05 (*).

Fig. 5

Effects of ketamine and ALCAR on MAO and COMT gene expression. The schematic diagram shows dopamine (DA), metabolized to DOPAC, 3-MT (3-Methoxytyramine) and HVA, by two enzymes, MAO and COMT (A). Embryos at 52 hpf were treated with lower doses of ketamine (0.1–0.3 mM) (B) as well as 2 mM ketamine, 2 mM ketamine plus 0.5 mM ALCAR or 0.5 mM ALCAR (C) alone for 24 h. Total RNA was extracted from the embryos (76 hpf) and after first strand cDNA synthesis from the RNA, quantitative real-time polymerase chain reaction (qRT-PCR) was performed. The 2-ΔΔCT method was used to determine relative gene expression for MAO and COMT. The GAPDH gene served as the internal control for all qRT-PCR experiments. Data were averaged and shown as normalized (fold-change over control) relative gene expression ± SDs. One-way ANOVA with comparison post-hocs were used to determine statistical significance with P = <0.05 (*).