Assessment of NMDA receptor NR1 subunit hypofunction in mice as a model for schizophrenia

Abstract

N-methyl-D-aspartate receptors (NMDARs) play a pivotal role in excitatory neurotransmission, synaptic plasticity and brain development. Clinical and experimental evidence suggests a dysregulation of NMDAR function and glutamatergic pathways in the pathophysiology of schizophrenia. We evaluated electrophysiological and behavioral properties of NMDAR deficiency utilizing mice that express only 5-10% of the normal level of NMDAR NR1 subunit. Auditory and visual event related potentials yielded significantly increased amplitudes for the P20 and N40 components in NMDAR deficient (NR1(neo)-/-) mice suggesting decreased inhibitory tone. Compared to wild types, NR1(neo)-/- mice spent less time in social interactions and showed reduced nest building. NR1(neo)-/- mice displayed a preference for open arms of a zero maze and central zone of an open field, possibly reflecting decreased anxiety-related behavioral inhibition. However, locomotor activity did not differ between groups in either home cage environment or during behavioral testing. NR1(neo)-/- mice displayed hyperactivity only when placed in a large unfamiliar environment, suggesting that neither increased anxiety nor non-specific motor activation accounts for differential behavioral patterns. Data suggest that NMDAR NR1 deficiency causes disinhibition in sensory processing as well as reduced behavioral inhibition and impaired social interactions. The behavioral signature in NR1(neo)-/- mice supports the impact of impaired NMDAR function in a mouse model with possible relevance to negative symptoms in schizophrenia.

Figure 1. Testing apparatus used to assess social behavior of NR1^neo-/- mice and wildtype littermates

(a) The behavioral testing apparatus viewed from above. There is a clear Plexiglas cylinder in each of the two end chambers. Before the start of each test, one of the end chambers was arbitrarily designated the “social side” (the side into which the stimulus mouse would be introduced), and the other end chamber was designated the “nonsocial side.” A “test” mouse is shown in the center chamber of the apparatus, and a “stimulus” mouse is shown in the cylinder on the social side of the apparatus (the “social cylinder”). The cylinder on the nonsocial side of the apparatus (the “nonsocial cylinder”) is empty. For the purposes of the picture, no lids are shown on the cylinders. (b) Multiple holes are evenly spaced over the surface of the cylinders in each end chamber, and these holes are large enough for a mouse to poke its nose through for olfactory investigation. A stimulus mouse is shown in the cylinder. (c) Dimensions of the behavioral testing apparatus. (Figure with permission from (Sankoorikal et al. 2006))

Figure 2. Increased auditory evoked brain activity in NR1^neo-/- mice

Following presentation of paired clicks, signal averaging was used to resolve the auditory event related potentials against the background of simultaneous brain activity. ERPs consist of different components with the P20 defined as the first major positive deflection (10-30ms) and the N40 defined as the following negative deflection (25-60ms). Traces in panel a and b show averaged first stimulus (S1, grey) and second stimulus responses (S2 black) for 16 NR1neo-/- and 15 WT mice (1 WT mouse lost the electrode pedestal during recording). The second response amplitude is decreased relative to the first at an interstimulus interval of 500ms, which allows for gating or habituation. This is also shown in panels c and d where S1 is significantly higher than S2 for NR1^neo-/- and WT mice. Statistical analysis of amplitudes yields a significant increase for both P20 and N40 amplitude in NR1^neo-/- mice compared to WT littermates (** p<0.01; *** p<0.001).

Figure 3. Increased visually evoked brain activity associated in NR1^neo-/- mice

Stimuli were presented as paired flashes; signal averaging was applied to resolve potentials against background brain activity. Visual ERPs consist of similar components as auditory-ERPs. Traces in panel a show grand average traces for S1 and S2 in NR1^neo-/- mice, traces in panel b are grand averages for WT mice. Amplitudes for NR1^neo-/- mice (7) were significantly larger than WT (8) for N1 (** p<0.01; *** p<0.001) while showing a trend towards significance for P1 (§ p=0.0597;). Mice which had seizures in previous tests (3 NR1^neo-/-) or lost their electrode pedestal before or during recording of visual ERPs (6 NR1^neo-/-, 8 WT mice) were excluded from statistical analysis.

Figure 4. Reduced sociability in NR1^neo-/- mice

Columns in panel a represent chamber preference scores, i.e. the amount of time mice spent in on the social side minus time spent on the non-social side of the 3-chambered social choice apparatus, in Phase 1A (stimulus mouse absent, 0-5min), Phase 1B (stimulus mouse absent, 5-10 min), and Phase 2 (stimulus mouse present, 10-15min). The social side refers to the side in which the stimulus mouse is placed. The non-social side refers to the side in which an inanimate object is placed. A negative value indicates a preference for the non-social side, whereas a positive value indicates a preference for the social side. NR1^neo-/- mice (N=7) showed markedly reduced time spent in social interaction relative to WT mice (N=12). Panel b illustrates the cylinder sniffing preference score, i.e. the time mice spent sniffing the social cylinder minus time spent sniffing the non-social cylinder in Phase 1A (stimulus mouse absent, 0-5min), Phase 1B (stimulus mouse absent, 5-10min), and Phase 2 (stimulus mouse present, 10-15min). Again, a negative value indicates a preference for sniffing the cylinder on the non-social side, whereas a positive value indicates a preference for sniffing the cylinder on the social side. WT mice show a significantly higher value in social sniffing during Phase 2 (stimulus mouse present, 10-15min) than NR1^neo-/- mice. The number of transitions between sectors (c) is comparable between genotypes, indicating that differences in locomotor activity do not account for differences in social choice measures shown in panels a and b (*** p<0.001). 3 NR1^neo-/- mice had a seizure while tested in the sociability apparatus and were removed from subsequent testing.

Figure 5. NR1^neo-/- mice spend more time in open arms of an elevated zero-maze

a) NR1^neo-/- mice (N=8) spent about 60% of the total time in the open sectors whereas WT (N=12) littermates stayed in the enclosed sectors and spent less than ~10% of the time in the open sectors. b) The number of entries into the open sectors shows no significant difference between genotypes. c) This significant difference cannot be attributed to different locomotor activity patterns as the distance covered by NR1^neo-/- and WT mice is comparable (***p<0.001). Three mice were excluded from analyses, two NR1^neo-/- mice jumped off the platform and one NR1^neo-/- mouse had a seizure during testing.

Figure 6. NR1^neo-/- mice spend more time in the central zone of the open field

Panels a, b, and c display results of testing in the open field paradigm (* p<0.05; *** p<0.001). As shown in panel a NR1^neo-/- mice (N=12) spent significantly more time in the central zone of the open field than WT mice (N=12). b) NR1^neo-/- mice also entered the central zone significantly more often than WT mice. c) These differences cannot be attributed to different locomotor activity because the distance covered by NR1^neo-/- and WT mice is comparable.

Figure 7. Locomotor activity in NR1^neo-/- mice is comparable to the locomotor activity of wildtype littermates in a familiar environment

Measurement of locomotor activity was performed with 2 cohorts of NR1^neo-/- and WT mice. Panel 7a illustrates a 30 minute comparison of locomotor activity in the home cage environment for the 1^st cohort. There was no significant effect of genotype on ambulation between NR1^neo-/-(N=7) and WT (N=10) mice (F(1,15)=0.834, p=0.7767). Mice which had seizures during previous tests (3 NR1^neo-/-) or lost the electrode pedestal (6 NR1^neo-/-, 6 WT mice) were excluded from testing. 7b illustrates the comparison of individual 5 minute time bins. Both mutants and wildtypes show the typical decrease in locomotor activity over time.

Figure 8. NR1^neo-/- mice display context dependent changes in locomotor activity

Results of the 1^st cohort led to a repetition with another cohort of mice (8 NR1^neo-/- and 8 WT littermates) at AstraZeneca Neuroscience (Wilmington, DE, USA). Comparison of locomotor activity under comparable conditions replicated the initial results (F(1,14)=1.0195, p=0.3298, as shown in panel 8a. Figure 8c) Placement in an unfamiliar environment (large cage without bedding) elicited an increase in locomotor activity in NR1^neo-/- mice relative to WT controls (** p<0.01). The recording time was extended to 120 minutes. Figure 8b) A shorter recording span did not detect this difference.