Expression levels of brain muscarinic acetylcholine receptor in offspring rats of drinking-water borne fluorosis

Abstract

Objective To observe the changes of learning and memory ability and detect the expression of muscarinic acetylcholine receptor(mAChR, M receptor) at mRNA and protein levels in brains of offspring rats with chronic fluorosis, and to reveal the mechanism of the central nervous system damage. Methods Forty healthy SD rats were divided into two groups (20 in each group, half male and half female) by random number table according to body weight. In the control group, the rats were fed with drinking water containing no more than 0.5 mg/L fluoride; in the fluoride group, the rats were fed with high dose of sodium fluoride in drinking water (50.0 mg/L fluoride). Each group was fed with normal diet (6.2 mg/kg fluoride). After exposed to fluoride for 6 months, each group was mated, and brains of newborn offspring rats aged 1, 7, 14, 21 and 28 days were taken, and expression of M1 and M3 receptors at mRNA and protein levels were analyzed by real-time PCR and Western blotting, respectively. Behavioral changes were measured by Morris water maze test at the 28 days after birth. The correlations between protein levels of M1 and M3 receptors and the ability of learning and memory at the 28 days after birth were analyzed. Results In fluoride group of the offspring rats at 28 days after birth, the escape latency time[(35.61±9.00)s] was significantly longer than that in control group [(8.46±3.09)s,P < 0.05], while the numbers of crossing the platforms and the time of staying the platforms [(5.00±2.90)times, (16.66±2.79)s] were significantly decreased as compared to that of control group [(15.17±3.66)times, (22.51±2.66)s, all P < 0.05]. Furthermore, the mRNA expression and the protein levels of M1 and M3 receptors in rat brain at each phase in fluoride group were significantly decreased as compared to controls [M1 mRNA in control groups: (100.00±11.00)%, (100.00±17.57)%, (100.00±9.14)%, (100.00±7.52)%, (100.00±15.78)%; M1 mRNA in fluoride groups: (20.47±8.07)%, (14.00±4.53)%, (16.57±7.62)%, (25.56±12.78)%, (16.27±4.82)%; M3 mRNA in control groups: (100.00±16.30)%, (100.00±14.40)%, (100.00±7.20)%, (100.00±14.31)%, (100.00±13.16)%; M3 mRNA in fluoride groups: (29.17±8.00)%, (12.77±2.22)%, (26.40±7.20)%, (15.74±3.55)%, (28.14±7.53)%; M1 protein in control groups: (100.00±2.24)%, (100.00±8.30)%, (100.00±4.61)%, (100.00±13.78)%, (100.00±11.72)%; M1 protein in fluoride groups: (20.47±8.07)%, (14.00±4.53)%, (16.57±7.62)%, (25.56±12.78)%, (16.27±4.82)%; M3 protein in control groups: (100.00±16.30)%, (100.00±14.40)%, (100.00±7.20)%, (100.00±14.31)%, (100.00±13.16)%; M3 protein in fluoride groups: (29.17±8.00)%, (12.77±2.22)%, (26.40±7.20)%, (15.74±3.55)%, (28.14±7.53)%, P < 0.05 or < 0.01]. The escape latency and M1, M3 receptors protein levels were negatively correlated (r =-0.827,-0.742, all P < 0.05), and the number of space exploration and M1, M3 receptors protein levels were positively correlated (r = 0.843, 0.806, all P < 0.05). Conclusion The expression of M receptor at protein and mRNA levels in offspring rat brains of different ages are significantly declined, which might be one of the mechanism of the decreased ability of learning and memory induced by fluoride toxicity. Key words: Fluorides; Brain; Muscarinic acetylcholine receptors; Learning; Memory; Rats