Β-N-Methylamino-L-Alanine (BMAA) Toxicity Is Gender and Exposure-Age Dependent in Rats

Abstract

Cyanobacterial β-N-methylamino-L-alanine (BMAA) has been suggested as a causative or contributory factor in the development of several neurodegenerative diseases. However, no BMAA animal model has adequately shown clinical or behavioral symptoms that correspond to those seen in either Alzheimer's Disease (AD), Amyotrophic Lateral Sclerosis (ALS) or Parkinson's Disease (PD). We present here the first data that show that when neonatal rats were exposed to BMAA on postnatal days 3, 4 and 5, but not on gestational day 14 or postnatally on days 7 or 10, several AD and/or PD-related behavioral, locomotor and cognitive deficits developed. Male rats exhibited severe unilateral hindlimb splay while whole body tremors could be observed in exposed female rats. BMAA-exposed rats failed to identify and discriminate a learned odor, an early non-motor symptom of PD, and exhibited decreased locomotor activity, decreased exploration and increased anxiety in the open field test. Alterations were also observed in the rats' natural passive defense mechanism, and potential memory deficits and changes to the rat's natural height avoidance behavior could be observed as early as PND 30. Spatial learning, short-term working, reference and long-term memory were also impaired in 90-day-old rats that had been exposed to a single dose of BMAA on PND 3-7. These data suggest that BMAA is a developmental neurotoxin, with specific target areas in the brain and spinal cord.

Keywords: BMAA; behavior; cognition; motor function; neurodegeneration; rat; rats; β-N-methylamino-L-alanine.

Figure 1

Percentage time rat dragged its tail, as observed in male and female Sprague Dawley rats (n = 30 for vehicle control and n = 10 for each other exposure age as indicated on the figure) during a ten-minute test period on PND 18. * Indicates significant difference to the control (p < 0.05).

Figure 2

Average nest finding times of male (A) and female (B) Sprague Dawley rats tested on PND 10. VC is vehicle control, G14 is exposure on gestation day 14. For the vehicle control n = 15 whereas n = 5 for all other groups. * Indicates significant difference to the control (p < 0.05).

Figure 3

Results of the modified Open Field Test in male (A,C,E,G) and female (B,D,F,H) Sprague Dawley rats. (A,B) shows the total number of lines crossed, (C,D)shows the number of entries into the center squares. (E,F) shows the total stationary time and (G,H) the number of rearing events. For each dose age and gender group n = 5, except for the vehicle control where n = 15. Significance (p < 0.05) is indicated by (*).

Figure 4

Average time spent in the open arms of the elevated plus maze observed in male (A) and female (B) Sprague Dawley rats and the number of repeated entries made by male (C) and female (D) rats into the open arms during the 10-min testing period as tested on PND 30. For the vehicle control n = 15, and n = 5 for all other groups as indicated on the graphs. * Indicates significant difference from the control (p < 0.05).

Figure 5

Duration Sprague Dawley rats (n = 30 for vehicle control and n = 10 for all other groups) were immobile after a 120 dB noise, following 5-min habituation to the test environment, as tested on PND 55 in the audiogenic freezing response test. * Indicates significant difference from the control (p < 0.05).

Figure 6

Reference memory errors observed in male Sprague Dawley rats (n = 15 for vehicle control and n = 5 for all other groups) exposed to BMAA on PND 3 (dotted bar), PND 4 (dark grey bar), PND 5 (horizontally striped bar), PND 6 (black bars) and PND 7 (diagonally striped-bars) compared to control (white bars) and vehicle control rats (light grey bars) in the acquisition trials (A) of the radial arm maze. In normal, healthy rats the observed reference memory errors will decrease with each consecutive acquisition trial as a function of learning. (B) Indicates the change in reference memory errors, or learning, over the five-day trial period observed in control (black line), vehicle control (grey line) and rats exposed to BMAA on PND 3 (black dashed line). Long-term memory, or retention of the learned task, allows the rats to remember the learned pattern on the radial arm maze. The reference memory errors observed in the retention test of the radial arm maze conducted one week after the last acquisition trial (C) compares the long-term memory of rats exposed to BMAA on PND 3–PND 7 compared to control rats. (*) Indicates significant difference from the control (p < 0.05).

Figure 7

Reference memory errors observed in female Sprague Dawley rats (n = 15 for vehicle control and n = 5 for all other groups) exposed to BMAA on PND 3 (dotted bars), PND 4 (dark grey bars), PND 5 (horizontally striped-bars), PND 6 (black bars) and PND 7 (diagonally striped-bars) compared to control (white bars) and vehicle control rats (light grey bars) in the acquisition trials (A) of the radial arm maze. In normal, healthy rats the observed reference memory errors will decrease with each consecutive acquisition trial as a function of learning. (B) Indicates the change in reference memory errors, or learning, over the five-day trial period observed in control (black solid line), vehicle control (grey line) and rats exposed to BMAA on PND 3 (black dashed line). Long-term memory, or retention of the learned task, allows the rats to remember the learned pattern on the radial arm maze. The reference memory errors observed in the retention test of the radial arm maze conducted one week after the last acquisition trial (C) compares the long-term memory of rats exposed to BMAA on PND 3–PND 7 compared to control rats. (*) Indicates significant difference from the control (p < 0.05).

Figure 8

Working memory errors observed in male (A) and female (B) Sprague Dawley rats neonatally exposed to 400 mg/kg BMAA compared to control and vehicle control rats. Errors across all acquisition trials were pooled (n = 75 for vehicle control and n = 25 for all other groups). (*) Indicate significant difference from the control (p < 0.05).

Figure 9

Recorded escape latencies, time to the platform, tested in the Morris water maze hidden platform test for control (white bar) and vehicle control rats (light grey bar) compared to rats exposed to 400 mg/kg BMAA on PND 3 (dotted bar), PND 4 (dark grey bar), PND 5 (horizontally striped bar), PND 6 (black bar) and PND 7 (diagonally striped bar) (n = 30 for vehicle control and n = 10 for all other groups). Normal, healthy rats learn to use spatial cues to navigate itself towards the hidden platform; they memorize this location and recall it in subsequent trials, and has progressively shorter latencies throughout the study (as seen for the control and vehicle control rats). (*) Indicates significant difference from the control (p < 0.05).

Figure 10

Percentage time spent in the correct quadrant, where the platform was located in the previous trials, in the probe trial (where platform is removed) of the Morris water maze observed in control, vehicle control and BMAA-treated Sprague Dawley rats (n = 30 for vehicle control and n = 10 for all other groups) as tested three days after the acquisition trials. (*) Indicates significant difference from the control (p < 0.05).

Figure 11

Experimental design timeline indicating ages of BMAA exposure together with the days behavioral, emotional response and cognitive tests listed below were conducted.