The longitudinal effects of early developmental cadmium exposure on conditioned place preference and cardiovascular physiology in zebrafish

Abstract

Cadmium (Cd) is a naturally occurring trace metal that is widely considered to be highly toxic to aquatic organisms and a significant health hazard to humans (Amzal et al., 2009; Bernhoft 2013; Burger, 2008; Satarug et al., 2009). The zebrafish (Danio rerio) has been used as a model organism for toxicological studies with Cd (Banni et al., 2011; Blechinger et al., 2007; Chow et al., 2009; Chow et al., 2008; Favorito et al., 2011; Kusch et al., 2007; Matz et al., 2007; Wang and Gallagher, 2013). We asked what the lasting longitudinal effects would be from short early developmental Cd exposure (between 24 and 96h post-fertilization) in a range that larvae might experience living atop typical Cd-containing surface sediments (0, 0.01, 0.1, 1.0 and 10μM CdCl₂: 1.124, 11.24, 112.4 and 1124μg Cd/L). The goal of this exposure window was to specifically target secondary neurogenesis, monoaminergic differentiation and cardiovascular development, without affecting earlier patterning processes. Developmental abnormalities in body size and CNS morphology increased with concentration, but were statistically significant only at the highest concentration used (10μM). Heart rate for Cd-treated larvae increased with concentration, and was significant even at the lowest concentration used (0.01μM). Longitudinal survival was significantly lower for fish developmentally exposed to the highest concentration. Except for brain weight, overall morphology was not affected by developmental Cd exposure. However, developmental exposure to lower concentrations of Cd (0.01, 0.1, and 1.0μM) progressively lowered cocaine-induced conditioned place preference (CPP), used to measure function of the reward pathways in the brain. Baseline heart rate was significantly lower in longitudinal fish developmentally exposed to 1.0μM Cd. Cardiovascular response to isoproterenol, a potent ß-adrenergic agonist, in longitudinal adults was also significantly affected by developmental exposure to Cd at low doses (0.01, 0.1 and 1.0μM). Surviving longitudinal adult fish exposed to the highest concentration of Cd showed normal CPP and cardiovascular physiology. The data imply that even lower exposure concentrations can potentially result in fitness-affecting parameters without affecting survival in a laboratory setting.

Keywords: Behavior; Cadmium; Cardiovascular; Morphology; Zebrafish.

Figure 1. The experimental design to examine the effects of Cd on early zebrafish development, longitudinal adult behavior, and physiology

The basic experimental design is shown in 1A. As described in methods, developing zebrafish were exposed to different concentrations of CdCl₂ starting at 24 hpf and ending at 96 hpf. After rinsing three times, the larvae were allowed 24 hours of recovery before imaging. Body and eye size were measured (1B). In addition, the areas of brain regions were determined (1C), including what is herein referred to as the telencephalon (T), diencephalon (D), and the hindbrain (H). Larvae from each treatment group were raised to adulthood and assessed for survival, sex, basic morphology, cardiovascular function using an electrocardiogram (ECG) and a conditioned place preference assay (CPP).

Figure 2. Cd decreases brain size and increases the number of acridine orange-positive (AO+) cells in the forebrain of treated larvae

Telencephalic area was significantly reduced in larvae treated with 10 μM CdCl₂, which is exemplified by comparing the brain in (2A) with that in (2B). While all three gross brain regions were reduced in size at this concentration, the most significant effect was seen for the telencephalon (2C, a: p < 0.001) and hindbrain (not shown, p < 0.001, see Table 1). The data from 6 experiments is shown in Figure 2C and in each of these the telencephalic area of Cd-treated larvae is expressed as a percentage of that from untreated controls in the same experiment (n = 73 for 0 μM, and n = 23-28 for the other concentrations, for statistical analysis the percentages were arcsin transformed). At least some of this effect was probably due to increased cell death as exemplified by comparing AO staining in panels (2D) and (2E). A significant change in the number of AO+ cells was seen at 10 μM of CdCl₂ but not at lower concentrations (Table 1, p < 0.001, n = 16 total for each Cd group from 3 experiments). Most of these cells were localized to the olfactory bulbs (asterisk in 2E), however, many were also found in the middle, ventral telencephalon (arrow in 2E). Many 10 μM Cd larvae had an arched appearance (2F). Scale bars in each panel represent 200 μm and the larva shown in 2F is 3.74 mm long.

Figure 3. Cd affects heart development and function

Cd caused a concentration-dependent increase in larval heart rate (3C, data was combined from 3 experiments with a total n = 20 for each Cd group, a: p < 0.01 compared to 0 μM, b: p< 0.001 when compared to 0 μM larvae, c: p < 0.05 when compared to 0.01 μM larvae, and d: p < 0.01 when compared to 0.01 μM larvae). Larvae treated with 10 μM CdCl₂ had more AO+ cells (small arrowheads in 2B and 2C) in the heart than untreated larvae. Most 10 μM Cd larvae had hearts that were normal in size and morphology (2D and 2E), but some had an enlarged ventricle and pericardium (3F). The asterisks highlight the ventricles, which showed no obvious defects in looping. There were no discernable differences in morphology or AO+ cells seen in larvae exposed to lower Cd concentrations (not shown). The scale bar for 2B and 2C is 200 μm, and that for 2D-2F is 150 μM.

Figure 4. Developmental Cd exposure affects survival and brain size in longitudinal adult fish

Fish treated with 10 μM CdCl₂ during development showed significantly lower survival at 6 months than untreated controls (4A). This experiment was carried out a minimum of 6 times for each treatment group (a: p < 0.05 when compared to the 0 μM, 0.1 μM and 1.0 μM groups and b: p < 0.01 compared to the 0.01 μM group). The telencephalons of longitudinal adult GFP⁺ fish were imaged and the area measured as described in methods (4B). Body length and telencephalic area are strongly correlated (3C, p < 0.0001, n = 152). The telencephalon to body length ratio (TA/BL) did not vary significantly between the different longitudinal treatment groups (4D this experiment was done 3 times with an n = 35 for each Cd group, except the 10 μM Cd, which had much lower survival and an n = 10, p < 0.52). However, the shape of the concentration response curve was similar to that for survival (4A) and larval telencephalic area (2C), with a slight, but statistically insignificant, increase at the lower concentrations (0.01 μM), and a decrease at higher concentrations (1.0 and 10 μM). Brain weight was strongly correlated with body length when all groups were considered together (3E, p < 0.0001, n = 103). The brain weight to body length ratio differed between CdCl₂ treatment groups. Fish treated with 0.1 μM, 1.0 μM and 10 μM CdCl₂ during development had proportionally heavier brains than untreated controls (n = 21-24 for each Cd group except the 10 μM group, which had an n =12, a: p < 0.01 compared to the 0 μM group, b: p < 0.001 compared to the 0 μM group, c: p < 0.01 when compared to the 0.01 μM group and d: p < 0.05 compared to the 1.0 μM fish).

Figure 5. Developmental Cd exposure affects CPP behavior and heart rate in longitudinal adult fish

Fish not treated with CdCl₂ during early development (0 μM Cd) and fish treated with 10 μM CdCl₂ display normal CPP in response to cocaine (**p < 0.01 when baseline trials are compared to post-conditioning trials with cocaine). The response of the 0 μM group is significantly higher than that of fish not conditioned with cocaine (5A Unt, a: p < 0.05). Developmental exposure to CdCl₂ progressively attenuated adult CPP, except at the highest concentration, 10 μM (5A b: p < 0.01 when compared to 0 μM Cd, c: p < 0.001 when compared to 0 μM CdCl₂ and p < 0.05 when compared to 10 μM Cd fish). This experiment was repeated 3 times, by different sets of investigators with a minimum total n = 30 for each group. Longitudinal adult fish were also tested for heart rate by measuring their ECG (Figure 5B). Adult fish treated with 1.0 μM CdCl₂ during development had a significantly lower baseline heart rate than fish from all other treatment groups (5B, a: p < 0.01 and b: p < 0.001 when compared to the 1 μM Cd fish). After determination of baseline heart rate, the same fish were tested for their cardiovascular response to isoproterenol, a potent β-adrenergic agonist. Fish not developmentally exposed to Cd (0 μM Cd) showed a significant response to the isoproterenol (** p = 0.01, when isoproterenol is compared to baseline using a paired t-test), as did the fish treated with 0.01 μM and 10 μM during development (*p < 0.05 and *** p < 0.001). In addition, fish treated with 0.01 μM CdCl₂ during development showed a significantly lower response to isoproterenol than either the 0 μM and 10 μM groups (a: p < 0.05 as determined by ANOVA of arcsin transformed values). This experiment was repeated twice, with ECG recordings by two sets of investigators with a minimum n = 8 for each Cd group.