Behavioral deficits and cholinergic pathway abnormalities in male Sanfilippo B mice

Abstract

Sanfilippo B syndrome is a progressive neurological disorder caused by inability to catabolize heparan sulfate glycosaminoglycans. We studied neurobehavior in male Sanfilippo B mice and heterozygous littermate controls from 16 to 20 weeks of age. Affected mice showed reduced anxiety, with a decrease in the number of stretch-attend postures during the elevated plus maze (p=0.001) and an increased tendency to linger in the center of an open field (p=0.032). Water maze testing showed impaired spatial learning, with reduced preference for the target quadrant (p=0.01). In radial arm maze testing, affected mice failed to achieve above-chance performance in a win-shift working memory task (t-test relative to 50% chance: p=0.289), relative to controls (p=0.037). We found a 12.4% reduction in mean acetylcholinesterase activity (p<0.001) and no difference in choline acetyltransferase activity or acetylcholine in whole brain of affected male animals compared to controls. Cholinergic pathways are affected in adult-onset dementias, including Alzheimer disease. Our results suggest that male Sanfilippo B mice display neurobehavioral deficits at a relatively early age, and that as in adult dementias, they may display deficits in cholinergic pathways.

Keywords: Acetylcholinesterase; Glycosaminoglycan; Inborn error of metabolism; Lysosomal; Mucopolysaccharidosis; Neurodegeneration.

Figure 1

Motor performance. (A) Rotarod testing in littermate controls (filled circles) and Sanfilippo B mice (open circles). RPM: revolutions per minute. (B) Open field locomotion testing (day 1). Experiment began in dark (shaded portion of graph), followed by light. Error bars represent SEM.

Figure 2

Anxiety/fear testing. (A) Proportion of time spent in the center of an open field during dark and light phases in littermate controls (filled circles) and Sanfilippo B mice (open circles). (B) Dot density plot of the number of stretch attend postures (a fear response) in Sanfilippo B and control mice during elevated plus maze testing. (C) Ratio of time spent interacting with a novel mouse to total time during phase 3 of the social interaction test. Error bars represent SEM. *p < 0.05.

Figure 3

Learning and memory. (A) Latency to find the hidden platform during Morris water maze testing in littermate controls (filled circles) and Sanfilippo B mice (open circles). Average of two trials per day is shown for days 1–4. Day 5 was a probe trial. (B) Time spent in the target quadrant during the first 30s in the Morris water maze. Symbols represent values of individual mice. Dashed lines represent group means. (C) Percent of choices that were incorrect during days 6–8 of phase 1 of radial arm maze testing. Bracket and asterisk indicate that control mice but not Sanfilippo B mice showed better than chance performance on the final day. (D) Across phase errors during days 6–8 of radial arm maze testing. Error bars represent SEM. *p < 0.05.

Figure 4

Acetylcholinesterase and choline acetyltransferase activity in brain. (A–B) Acetylcholinesterase (AChE) activity staining (brown) in coronal sections of (A) control and (B) Sanfilippo B mice (representative of n = 3 per group). AG: amygdala; H: hippocampus. (C–H) Enzymatic activity of AChE and choline acetyltransferase (ChAT) and acetylcholine levels in (C–E) whole brain homogenates and (F–H) coronal section 3 of the brain. Box plots show median, 25^th and 75^th centiles, and whiskers represent 5^th and 95^th centiles. Dot plots show values for individual mice. Dashed lines show group means. *p < 0.05.