Antipsychotic drugs disrupt normal development in Caenorhabditis elegans via additional mechanisms besides dopamine and serotonin receptors

Abstract

Antipsychotic drugs may produce adverse effects during development in humans and rodents. However, the extent of these effects has not been systematically characterized nor have molecular mechanisms been identified. Consequently, we sought to evaluate the effects of an extensive panel of antipsychotic drugs in a model organism, Caenorhabditis elegans, whose development is well characterized and which offers the possibility of identifying novel molecular targets. For these studies, animals were grown from hatching in the presence of vehicle (control) or antipsychotic drugs over a range of concentrations (20-160microM) and growth was analyzed by measuring head-to-tail length at various intervals. First-generation antipsychotics (e.g., fluphenazine) generally slowed growth and maturation more than second-generation drugs such as quetiapine and olanzapine. This is consistent with in vitro effects on human neuronal cell lines. Clozapine, a second-generation drug, produced similar growth deficits as haloperidol. Converging lines of evidence, including the failure to rescue growth with high concentrations of agonists, suggested that the drug-induced delay in development was not mediated by the major neurotransmitter receptors recognized by the antipsychotic drugs. Moreover, in serotonin-deficient tph-1 mutants, the drugs dramatically slowed development and led to larval arrest (including dauer formation) and neuronal abnormalities. Evaluation of alternative targets of the antipsychotics revealed a potential role for calmodulin and underscored the significance of Ca(2+)-calmodulin signaling in development. These findings suggest that antipsychotic drugs may interfere with normal developmental processes and provide a tool for investigating the key signaling pathways involved.

Fig. 1

First-generation antipsychotic drugs affect developmental growth in C. elegans. Differential interference contrast (DIC) images (100X) of animals grown for 72 hr in the presence of vehicle (control, left panels), or drugs (right panels): (A) chlorpromazine (160 μM), (C) fluphenazine (160 μM), or (E) haloperidol (160 μM). In addition, head-to-tail body length was analyzed in N2 animals at 24, 48, and 72 hr (N=25 at each time point/condition) treated with vehicle (DMSO) or (B) chlorpromazine, (D) fluphenazine, or (F) haloperidol at 20, 40, 80, and 160 μM as indicated in the legends. The data represent the means and standard deviations (error bars) at each time interval.

Fig. 2

Second-generation antipsychotic drugs produce modest effects on development in C. elegans. The data are presented according to the same scheme as Fig. 1. Drugs and the concentrations tested are indicated in the legends.

Fig. 3

Comparison of antipsychotic drugs that slow growth in C. elegans. Growth was measured as a function of body length in tph-1(mg280) and N2 animals treated with vehicle (control), butaclamol, metergoline, clozapine, haloperidol, fluphenazine, or chlorpromazine at (A) 24, (B) 48, and (C) 72 hr after hatching onto drug plates. All drugs were tested at the same concentration (160 μM). The data represent the means and standard deviations calculated from 25 animals per group. The conditions that are listed in the legend from top to bottom correspond to the bars presented from left to right for this and subsequent figures.

Fig. 4

Control experiments for biological effects of antipsychotic drugs. (A, B) First-generation and second-generation antipsychotic drugs were evaluated at the concentrations indicated for their ability to inhibit paralysis in C. elegans induced by 15 mM dopamine. In addition, the dopamine receptor agonist, quinpirole, was tested as a positive control for the paralytic effects of dopamine. For these experiments, adult N2 animals were preincubated with drugs or solvent (control) for 20 min prior to their introduction to test plates containing dopamine and drug or solvent. Animals (25–35 per plate) were examined for paralysis over time and the experiment was performed in triplicate. The data represent the averages from the 3 plates and are expressed as the fraction of immobile animals. All of the antipsychotic drugs produced significant inhibition of dopamine effects at all time points. (C) Pharyngeal pumping was measured in control (DMSO) or drug-treated animals (clozapine, olanzapine, haloperidol, or chlorpromazine at the concentrations indicated) at the 4^th larval stage (L4) by counting the number of pharyngeal pumps over 30 sec. Duplicate plates were tested for each condition and twenty animals were analyzed from each plate (N=40). Significant differences from the control group are indicated by asterisks: **p < 0.01.

Fig. 5

Effects of antipsychotic drugs on development in relation to serotonergic and dopaminergic systems. (A) tph-1 mutants were grown in the presence of vehicle (DMSO), clozapine (200 μM), or fluphenazine (200) μM at 20 °C and photographed (60X magnification) on the fourth day after hatching. (B) Quantitative analysis of the developmental staging (Stage %) of animals classified as adults, arrested, or dauer/partial dauer larvae (refer to Methods for criteria) on the third and fourth day after hatching. (C) Body length was measured for N2 animals treated with vehicle (DMSO), serotonin (2.5 or 5 mM), clozapine (160 μM), and clozapine (160 μM) with serotonin (2.5 or 5 mM) at 24, 48, and 72 hr after hatching onto plates. (D) In a similar manner, N2 animals were grown in the presence of vehicle (DMSO), quinpirole (750 μM or 1mM), clozapine (160 μM), fluphenazine (160 μM), clozapine with quinpirole (750 μM or 1 mM), and fluphenazine (160 μM) with quinpirole (750 μM or 1 mM) and body length was measured 72 hr after hatching. Each data point represents the average of 25 animals per condition. Error bars represent standard deviations from the mean. Statistical significance is indicated as before.

Fig. 6

Effect of calmodulin inhibitors on development in C. elegans. (a) Body length (μm) was measured and averaged from 25 animals that were grown in the presence of vehicle (control), selective calmodulin inhibitors (W-13 and calmidazolium), or antipsychotic drugs (chlorpromaize, fluphenazine, and trifluoperazine) for 72 hr after hatching. All compounds were tested at 80 and 160 μM. Error bars represent the standard deviations from the mean. Statistical differences from the control group are indicated as before. (B) Linear regression analysis of the potency of antipsychotic drugs as calmodulin antagonists (x-axis) vs. their effects on growth (body length) in C. elegans. This analysis was limited to drugs that were pharmacologically characterized as calmodulin antagonists and included, trifluoperazine, fluphenazine, chlorpromazine, haloperidol, and clozapine. The body length data were obtained from head-to-head comparisons of the drugs at the 72 hr time point. The potency (IC₅₀) data were compiled from different sources and refer to inhibition of functional activity of calmodulin [45,46].