Developmental lead exposure causes startle response deficits in zebrafish

Abstract

Lead (Pb(2+)) exposure continues to be an important concern for fish populations. Research is required to assess the long-term behavioral effects of low-level concentrations of Pb(2+) and the physiological mechanisms that control those behaviors. Newly fertilized zebrafish embryos (<2h post fertilization; hpf) were exposed to one of three concentrations of lead (as PbCl(2)): 0, 10, or 30 nM until 24 hpf. (1) Response to a mechanosensory stimulus: Individual larvae (168 hpf) were tested for response to a directional, mechanical stimulus. The tap frequency was adjusted to either 1 or 4 taps/s. Startle response was recorded at 1000 fps. Larvae responded in a concentration-dependent pattern for latency to reaction, maximum turn velocity, time to reach V(max) and escape time. With increasing exposure concentrations, a larger number of larvae failed to respond to even the initial tap and, for those that did respond, ceased responding earlier than control larvae. These differences were more pronounced at a frequency of 4 taps/s. (2) Response to a visual stimulus: Fish, exposed as embryos (2-24 hpf) to Pb(2+) (0-10 μM) were tested as adults under low light conditions (≈ 60 μW/m(2)) for visual responses to a rotating black bar. Visual responses were significantly degraded at Pb(2+) concentrations of 30 nM. These data suggest that zebrafish are viable models for short- and long-term sensorimotor deficits induced by acute, low-level developmental Pb(2+) exposures.

Figure 1

Concentration-dependent uptake of Pb²⁺ in whole embryo during 2–24 hours post fertilization (n = 100 embryos/sample; 3 samples/concentration). Values = mean ± SE. * = P < 0.05; ** = P < 0.01

Figure 2

Time- and concentration-dependent larval response to repeated stimuli at a frequency of 1 tap/sec. ▮= 0 nM Pb²⁺; ▯= 10 nM Pb²⁺; = 30 nM Pb²⁺. Values = proportional number of larvae responding at each stimulus ± SD. Interval between each stimulus = 1000 msec. n = 10/exposure concentration. * = P < 0.0001 relative to control value at equivalent stimulus time.

Figure 3

Responses to each stimulus at a frequency of 1 tap/sec. ▮= 0 nM Pb²⁺; ▯= 10 nM Pb²⁺; = 30 nM Pb²⁺. A. Time (msec) to initial response (reaction latency); B. Time (msec) to reach maximum head turning velocity (V_max); C. V_max (body lengths/sec); D. Duration (msec) of response (startle response + escape swim). Values = mean ± SE. Interval between each stimulus = 1000 msec. n = 10/exposure concentration. * = P < 0.05; ** = P < 0.01; *** = P < 0.001 relative to control value at equivalent stimulus time.

Figure 3

Responses to each stimulus at a frequency of 1 tap/sec. ▮= 0 nM Pb²⁺; ▯= 10 nM Pb²⁺; = 30 nM Pb²⁺. A. Time (msec) to initial response (reaction latency); B. Time (msec) to reach maximum head turning velocity (V_max); C. V_max (body lengths/sec); D. Duration (msec) of response (startle response + escape swim). Values = mean ± SE. Interval between each stimulus = 1000 msec. n = 10/exposure concentration. * = P < 0.05; ** = P < 0.01; *** = P < 0.001 relative to control value at equivalent stimulus time.

Figure 3

Responses to each stimulus at a frequency of 1 tap/sec. ▮= 0 nM Pb²⁺; ▯= 10 nM Pb²⁺; = 30 nM Pb²⁺. A. Time (msec) to initial response (reaction latency); B. Time (msec) to reach maximum head turning velocity (V_max); C. V_max (body lengths/sec); D. Duration (msec) of response (startle response + escape swim). Values = mean ± SE. Interval between each stimulus = 1000 msec. n = 10/exposure concentration. * = P < 0.05; ** = P < 0.01; *** = P < 0.001 relative to control value at equivalent stimulus time.

Figure 3

Responses to each stimulus at a frequency of 1 tap/sec. ▮= 0 nM Pb²⁺; ▯= 10 nM Pb²⁺; = 30 nM Pb²⁺. A. Time (msec) to initial response (reaction latency); B. Time (msec) to reach maximum head turning velocity (V_max); C. V_max (body lengths/sec); D. Duration (msec) of response (startle response + escape swim). Values = mean ± SE. Interval between each stimulus = 1000 msec. n = 10/exposure concentration. * = P < 0.05; ** = P < 0.01; *** = P < 0.001 relative to control value at equivalent stimulus time.

Figure 4

Time- and concentration-dependent larval response to repeated stimuli at a frequency of 4 taps/sec. A. ▮= 0 nM Pb²⁺; B. ▯= 10 nM Pb²⁺; C. = 30 nM Pb²⁺. Values = proportional number of larvae responding at each tap stimulus ± SD. Interval between each stimulus = 250 msec. n = 10/exposure concentration. * = P < 0.05; ** = P < 0.01; *** = P <0.005 relative to control value at equivalent stimulus time.

Figure 4

Time- and concentration-dependent larval response to repeated stimuli at a frequency of 4 taps/sec. A. ▮= 0 nM Pb²⁺; B. ▯= 10 nM Pb²⁺; C. = 30 nM Pb²⁺. Values = proportional number of larvae responding at each tap stimulus ± SD. Interval between each stimulus = 250 msec. n = 10/exposure concentration. * = P < 0.05; ** = P < 0.01; *** = P <0.005 relative to control value at equivalent stimulus time.

Figure 4

Time- and concentration-dependent larval response to repeated stimuli at a frequency of 4 taps/sec. A. ▮= 0 nM Pb²⁺; B. ▯= 10 nM Pb²⁺; C. = 30 nM Pb²⁺. Values = proportional number of larvae responding at each tap stimulus ± SD. Interval between each stimulus = 250 msec. n = 10/exposure concentration. * = P < 0.05; ** = P < 0.01; *** = P <0.005 relative to control value at equivalent stimulus time.

Figure 5

Representative traces of movement of larvae developmentally exposed to 0, 10 or 30 nM Pb²⁺ in response to a directional stimulus at a frequency of 4 taps/sec for 2.25 seconds. Interval between each stimulus = 250 msec.

Figure 6

Responses to each stimulus at a frequency of 4 taps/sec. ▮= 0 nM Pb²⁺; ▯= 10 nM Pb²⁺; = 30 nM Pb²⁺. A. Time (msec) to initial response (reaction latency); B. Time (msec) to reach maximum head turning velocity (V_max); C. V_max (body lengths/sec); D. Duration (msec) of response (startle response + escape swim). Values = mean ± SE. Interval between each stimulus = 250 msec. n = 10/exposure concentration. * = P < 0.05; ** = P < 0.01; *** = P <0.005 relative to control value at equivalent stimulus time.

Figure 6

Responses to each stimulus at a frequency of 4 taps/sec. ▮= 0 nM Pb²⁺; ▯= 10 nM Pb²⁺; = 30 nM Pb²⁺. A. Time (msec) to initial response (reaction latency); B. Time (msec) to reach maximum head turning velocity (V_max); C. V_max (body lengths/sec); D. Duration (msec) of response (startle response + escape swim). Values = mean ± SE. Interval between each stimulus = 250 msec. n = 10/exposure concentration. * = P < 0.05; ** = P < 0.01; *** = P <0.005 relative to control value at equivalent stimulus time.

Figure 6

Responses to each stimulus at a frequency of 4 taps/sec. ▮= 0 nM Pb²⁺; ▯= 10 nM Pb²⁺; = 30 nM Pb²⁺. A. Time (msec) to initial response (reaction latency); B. Time (msec) to reach maximum head turning velocity (V_max); C. V_max (body lengths/sec); D. Duration (msec) of response (startle response + escape swim). Values = mean ± SE. Interval between each stimulus = 250 msec. n = 10/exposure concentration. * = P < 0.05; ** = P < 0.01; *** = P <0.005 relative to control value at equivalent stimulus time.

Figure 6

Responses to each stimulus at a frequency of 4 taps/sec. ▮= 0 nM Pb²⁺; ▯= 10 nM Pb²⁺; = 30 nM Pb²⁺. A. Time (msec) to initial response (reaction latency); B. Time (msec) to reach maximum head turning velocity (V_max); C. V_max (body lengths/sec); D. Duration (msec) of response (startle response + escape swim). Values = mean ± SE. Interval between each stimulus = 250 msec. n = 10/exposure concentration. * = P < 0.05; ** = P < 0.01; *** = P <0.005 relative to control value at equivalent stimulus time.

Figure 7

Effect of developmental Pb²⁺ exposure on the adult expression of visual responses to a rotating black bar (10 revolutions/min) under low light conditions (N = 12/exposure concentration). Bars (mean ± SE) with different letters are statistically different from mean control response (ANOVA, P< 0.001, Student-Newman-Keuls post hoc test, P < 0.05). Means with the same letter are not statistically different.