Neurocognitive and psychotiform behavioral alterations and enhanced hippocampal long-term potentiation in transgenic mice displaying neuropathological features of human alpha-mannosidosis

Abstract

Mice with alpha-mannosidase gene inactivation provide an experimental model for alpha-mannosidosis, a lysosomal storage disease with severe neuropsychological and psychopathological complications. Neurohistological alterations in these mice were similar to those in patients and included vacuolations and axonal spheroids in the CNS and peripheral nervous system. Vacuolation was most prominent and evenly distributed in neuronal perikarya of the hippocampal CA2 and CA3 regions, whereas CA1 and dentate gyrus were weakly or not affected. Field potential recordings from CA1 region in hippocampal slices showed enhanced theta burst-induced long-term potentiation (LTP) in alpha-mannosidase-deficient mice. Longitudinal assessment in age-matched alpha-mannosidase-deficient and wild-type littermates, using an extended test battery, demonstrated a neurocognitive and psychotiform profile that may relate to the psychopathological alterations in clinical alpha-mannosidosis. Brainstem auditory-evoked potentials and basic neuromotor abilities were not impaired and did not deteriorate with age. Exploratory and conflict tests revealed consistent decreases in exploratory activity and emotional blunting in the knock-out group. alpha-Mannosidosis mice were also impaired in aversively motivated learning and acquisition of signal-shock associations. Acquisition and reversal learning in the water maze task, passive avoidance learning in the step-through procedure, as well as emotional response conditioning in an operant procedure were all impaired. Acquisition or shaping of an appetitive instrumental conditioning task was unchanged. Appetitive odor discrimination learning was only marginally impaired during shaping, whereas both the discrimination and reversal subtasks were normal. We propose that prominent storage and enhanced LTP in hippocampus have contributed to these specific behavioral alterations in alpha-mannosidase-deficient mice.

Figure 1.

Light micrographs demonstrate lysosomal storage in the CNS of α-mannosidase-deficient mice. Micrographs show the hippocampal CA3 region (A, B) and lamina 5/6 neocortex (C, D) in 2-month-old wild-type (A, C) and α-mannosidase-deficient (B, D) mice. In the hippocampal CA3 region (B), most neuronal perikarya show clear cytoplasmic vacuoles that are the morphological equivalent for lysosomal storage of mannose-rich oligosaccharides. In the neocortex (D), vacuolation of neuronal perikarya (arrows) is more variable. Semithin sections, Toluidine blue staining. Scale bar, 20 μm.

Figure 2.

Hippocampal long-term potentiation in 3-month-old wild-type and α-mannosidase-deficient mice. α-Mannosidase-deficient mice (open circles) displayed marginally stronger LTP than wild-type littermates (filled circles), when the potentiation was induced by a single HFS but marked enhancement of potentiation in response to TBS (10 burst of 4 stimuli at 100 Hz). Both groups generated a similar decremental LTP after HFS, but knock-outs still showed a significant potentiation after 120 min (p < 0.05, Wilcoxon's test). Knock-out mice displayed a higher initial LTP magnitude after TBS and maintained this difference throughout the recording (p < 0.05, repeated-measures ANOVA). Graphs show mean slopes of fEPSP and SEMs (error bars), expressed as a percentage of initial value.

Figure 3.

Electron micrographs of spiral ganglia and auditory-evoked potentials in wild-type and α-mannosidase-deficient mice. Electron micrographs of spiral ganglia in a 6-month-old wild-type (A) and a 2-month-old α-mannosidase-deficient (B) mouse demonstrate lysosomal storage in the deficient animal. Two vacuoles from a spiral ganglion cell at a higher resolution show the limiting membrane and the low amounts of granular contents (C). Most of the storage material is artificially leached during tissue preparation. Scale bars: A, B, 10 μm; C, 0.5 μm. Brainstem auditory-evoked potentials, conversely, show no difference between wild-type and α-mannosidase-deficient mice at either age (D). Examples shown in this figure are from a 3-month-old control and knock-out mouse (left and right ears). The table at the bottom of the figure shows mean latency for each of the five peaks that were identified in the left or right ear recordings of the tested mice.

Figure 4.

Mean home cage activity during a 24 h recording session in 3- and 12-month-old wild-type and α-mannosidase knock-out mice. The exploratory peak recorded during introduction of animals into the cages was slightly lower in knock-out mice (open circles), but overall activity was not significantly different between the groups.

Figure 5.

Open-field locomotion in 3- and 12-month-old wild-type and α-mannosidase knock-out mice. Bars indicate mean values and SEM (error bars) in mice at 3 months (black bars) and 12 months (white bars) of age. Both path length and corner crossings are significantly reduced in the knock-out group (^*p < 0.05).

Figure 6.

Water maze performance in 3-month-old wild-type and α-mannosidase knock-out mice. Acquisition of the task (top part of the figure) consisted of 10 trial blocks with a probe trial after each five blocks (probe 1 and probe 2). The escape platform remained in the same position on all acquisition trial blocks. Plots show mean escape latency (and SEM) for wild-type (filled circles) and knock-out (open circles) mice. Escape latency was consistently longer in the knock-outs on all trials blocks, but differences were smaller toward the end of training. Bottom part of the figure shows mean percentage of time (and SEM) spent in each of the four pool quadrants (i.e., target, adjacent 1 and 2, and opposite quadrant) during probe trial 1 and 2. Knock-outs search significantly less in the target quadrant (black bars) during probe 1 (^**p < 0.01) but not during probe 2. However, knock-outs still spent equal amounts of time in the target and opposite quadrant during probe 2.

Figure 7.

Water maze training continued in 12-month-old wild-type and α-mannosidase knock-out mice. Acquisition of the task (top part of the figure) consisted of 10 trial blocks with a probe trial after each five blocks (probe 1 and probe 2). The escape platform was in the same position as during the 3-month testing during trial blocks 1-5 but was changed to the opposite quadrant during blocks 6-10 (reversal trials). Plots show mean escape latency (and SEM) for wild-type (filled circles) and knock-out (open circles) mice. Escape latency was now only slightly longer in the knock-outs. The bottom part of the figure shows mean percentage (and SEM) of time spent in each of the four pool quadrants (i.e., target, adjacent 1 and 2, and opposite quadrant) during probe trials 1 and 2. Knock-outs search significantly less in the target quadrant (black bars) during probe 1 (^**p < 0.01) but not during probe 2. Again, knock-outs spent equal amounts of time in the target and opposite quadrant during probe 2.

Figure 8.

Odor discrimination conditioning in 3-month-old (A) and 12-month-old (B) wild-type and α-mannosidase knock-out mice. Bars depict mean preference (and SEM) in wild-type (black bars) and knock-out (white bars) mice expressed as percentage of time spent digging in the target vial. The initial discrimination trials were less consistent in the knock-outs (open circles in preference plot, A), but additional training eventually stabilized their performance. Overall discriminatory and reversal performances were similar in both genotype groups (bar charts in A and B). C, Controls; KO, knock-outs.

Figure 9.

Appetitive conditioning and CER trials in 12-month-old wild-type and α-mannosidase knock-out mice. During the initial training (A), response rates (expressed as mean number of nose pokes per minute) gradually increased, but rates were not significantly different between wild-type (filled circles) and α-mannosidase knock-out (open circles) mice. High response rates were displayed during the last training trials with intermittent food reinforcement. During 12 CER trials (B), tone-and-shock presentations (during unconditioned emotional responding trials) and tone presentations (during conditioned emotional responding trials) are superimposed on the intermittently reinforced conditioning schedule. Response rates are severely suppressed during the CER trials, but mean response rate is consistently higher in the knock-out group during all CER trials.