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, 6 (4), e778

Knockout of NMDA-receptors From Parvalbumin Interneurons Sensitizes to Schizophrenia-Related Deficits Induced by MK-801

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Knockout of NMDA-receptors From Parvalbumin Interneurons Sensitizes to Schizophrenia-Related Deficits Induced by MK-801

A M Bygrave et al. Transl Psychiatry.

Abstract

It has been suggested that a functional deficit in NMDA-receptors (NMDARs) on parvalbumin (PV)-positive interneurons (PV-NMDARs) is central to the pathophysiology of schizophrenia. Supportive evidence come from examination of genetically modified mice where the obligatory NMDAR-subunit GluN1 (also known as NR1) has been deleted from PV interneurons by Cre-mediated knockout of the corresponding gene Grin1 (Grin1(ΔPV) mice). Notably, such PV-specific GluN1 ablation has been reported to blunt the induction of hyperlocomotion (a surrogate for psychosis) by pharmacological NMDAR blockade with the non-competitive antagonist MK-801. This suggests PV-NMDARs as the site of the psychosis-inducing action of MK-801. In contrast to this hypothesis, we show here that Grin1(ΔPV) mice are not protected against the effects of MK-801, but are in fact sensitized to many of them. Compared with control animals, Grin1(ΔPV)mice injected with MK-801 show increased stereotypy and pronounced catalepsy, which confound the locomotor readout. Furthermore, in Grin1(ΔPV)mice, MK-801 induced medial-prefrontal delta (4 Hz) oscillations, and impaired performance on tests of motor coordination, working memory and sucrose preference, even at lower doses than in wild-type controls. We also found that untreated Grin1(ΔPV)mice are largely normal across a wide range of cognitive functions, including attention, cognitive flexibility and various forms of short-term memory. Taken together these results argue against PV-specific NMDAR hypofunction as a key starting point of schizophrenia pathophysiology, but support a model where NMDAR hypofunction in multiple cell types contribute to the disease.

Conflict of interest statement

PJH received consulting fees from Boehringer Ingelheim, Sunovion and Hoffman-La Roche Ltd, honorarium as Deputy Editor of Biological Psychiatry, lecture fees from Otsuka and fees for serving as an expert witness and consultant on patent litigation cases. DMK received honorarium as Editor of Brain. The remaining authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schizophrenia-related endophenotypes and cognitive function in Grin1ΔPV mice. (a and b) Novelty-induced hyperlocomotion: (a) Average beam-break counts over 2 h aggregated in 5-min bins for cohorts of 2 months (left) and 5 months (right) age (Error bars: s.e.m.). (b) Average total beam breaks for both age groups as indicated. Different age groups are distinct cohorts. (c) Average pre-pulse inhibition expressed as % for the individual dB-levels of pre-pulse. (d) Average absolute response to the startle-pulse (120 dB) at start and end of the test sequence, showing habituation to the startle-pulse over time. (e) Sucrose preference for 3 consecutive days during which two water bottles were presented in the first day, and one water and one bottle with 10% sucrose solution was offered on 2 consecutive days. Line graphs (left axis) represent the preference for the bottle that contained sucrose on days 2–3, while bar graphs show the absolute consumption of water (light blue/red) and sucrose (dark blue/red; right axis). (f) Short-term memory for spatial, non-spatial or social stimuli as indicated, assessed by spatial novelty preference (Y-maze), novel-object recognition and the three-chamber social memory task, respectively. Preference scores were calculated as the total time spent with the novel stimulus divided by the time spent with both stimuli combined. The yellow line indicates equal preference (chance levels). (g) Spatial working memory: correct choices (alternation of choice arm from sample to choice run) in the rewarded alternation paradigm in the T-maze are shown for training days 1–3 with 5-s intra-trial interval (ITI), and test days with 1 s and 15 s ITI. ITI-numbers are written in black to indicate round-robin and gray for massed trails. The same cohort was trained and tested twice at 2 months (left) and 6 months (right) of age. The yellow line indicates chance level. (h and i) Spatial reference memory and cognitive flexibility: (h) Percent of correct choices in the previous 20 trials in the plus maze plotted in intervals of 5 trials during acquisition and reversal learning. (i) Average number of blocks needed to reach criterion (85% correct in the previous 20 trials). (j–n) Averaged measures from the first 2 sessions in each of the 6 training stages mice were taken through in the 5-CSRTT (see Supplementary Methods for details): (j) % accuracy, (k) % correct responses, (l) % perseverative responses, (m) % premature responses and (n) reward latency. Blue, controls; red, knockouts. Asterisks indicate significant differences at P<0.05 measured by the appropriate statistical test (see Supplementary Table S1 for details). Error bars indicate 95% confidence intervals except were indicated otherwise. Ctrl, control; KO, knockout.
Figure 2
Figure 2
Effect of MK-801 on locomotor activity. (a) Dose–response curve of total beam breaks in 90 min post injection for rising doses of MK-801 (0.025 to 0.4 mg kg−1, n=4 per dose) in male C57bl6 mice. (b) Knockout mouse in MK-801-induced catalepsy (see also Supplementary Videos 2, 3, 4). (c–e) Total beam breaks for 60 min post injection for 0.1 (c), 0.2 (d) and 0.5 (e) mg kg−1 MK-801 vs vehicle. Asterisks indicate significant differences between MK-801-injected groups and relevant control groups (P<0.05, ANOVA). Note that beam-break counts in vehicle-injected KO mice are significantly higher compared with vehicle-injected controls in all three panels (indication omitted for clarity). (f–h) Average number of beam breaks over 30 min before and 90 min after injection of vehicle/MK-801 in 5-min intervals. (i) Total beam breaks for 60–90 min post injection of 0.1 (left), 0.2 (middle) and 0.5 (right) mg kg−1 MK-801 vs vehicle. Annotation as in c–e. (j and k) Counts for the three principle categories of behavior induced by 0.2 (j) and 0.5 (k) mg kg−1 MK-801: catalepsy, stereotypies and ataxia as indicated (see Results for details of scoring) for each animal expressed as % of total experimental time post injection (90 min). Solid circles, males; open circles, females. Asterisks indicate significant pair-wise differences (P<0.05, MW U-test or t-test as appropriate). (l–n) % time spent in states of catalepsy (l), stereotypies (m) and ataxia (n) plotted in 5-min intervals for 90 min after injection of 0.2 (left) or 0.5 (right) mg kg−1 MK-801. (o) Total beam-break counts over 90 min after injection of 0.2 mg kg−1 amphetamine (dark blue, dark red) or vehicle (light blue, light red). Asterisks indicate significant differences between drug-injected groups and relevant control groups (P<0.05, ANOVA). Error bars represent s.e.m. in line graphs (fh) and otherwise 95% confidence intervals. All experiments were conducted as between-subjects designs. MK-801 (dark blue, dark red) or vehicle (light blue, light red) indicated in control (blue) and knockout (red) mice. Gray numbers represent doses of MK-801 for each panel. ANOVA, analysis of variance; Ctrl, control; KO, knockout; NS, not significant.
Figure 3
Figure 3
MK-801-induced impairment of coordination, working memory and sucrose preference. (a–c) MK-801-induced impairment of motor coordination on the rotarod. (a and b) Time the males (a) or females (b) managed to remain on the accelerating rotarod at 30 min (left) and 60 min (right) after the injection of MK-801 (dark blue, dark red; 0.15 mg kg−1 in females; 0.2 mg kg−1 in males) or vehicle (light blue, light red). Asterisks indicate significant differences at P<0.05 (ANOVA). (c) The total number of failures (falling of the rod before acceleration) on three attempts at 60 min after injection (color code as in a). Asterisks indicate significant differences at P<0.05 (MW U-test). (d) Correct choices (% out of 10 trials per block) on the T-maze rewarded alternation task are shown for the first three blocks of training, as well as for MK-801- and matched vehicle-trials at increasing dose of MK-801 as indicated. The asterisks indicate a significant drug–genotype interaction at 0.15 and 0.2 mg kg−1 (only knockouts are affected), or a significant effect of drug at 0.4 mg kg−1 (both genotypes are affected); repeated measures ANOVA followed by t-tests. (e) Latencies on the choice trials (measured from insertion into the start arm until arrival in the choice arm to an accuracy of 1 s, x-axis) vehicle (left) and corresponding MK-801 (right) trials are plotted for every trial of each animal and dose (vertical axis). Each animal successfully ran 10 out of the 10 trials in each block, but data points from trials with identical latencies are plotted on top of one another. (f) Percent sucrose-solution (1%) consumption of total liquid consumption is plotted for 2 habituation days (left), as well as for vehicle (V)- and MK-801 (MK)-trials at the indicated doses (in mg kg−1). The asterisks indicate significant drug–genotype interaction at 0.1 mg kg−1 (only knockouts are affected), or a significant effect of drug at 0.15 mg kg−1 (both genotypes are affected); repeated measures ANOVA followed by t-tests. In all plots blue lines represent controls, red lines knockouts, with MK-801 (dark blue, dark red) or vehicle (light blue, light red) as indicated. Yellow lines indicate chance levels (50%). Error bars represent 95% confidence intervals. All experiments were conducted as within-subjects designs. ANOVA, analysis of variance; Ctrl, control; KO, knockout.
Figure 4
Figure 4
Induction of medial-prefrontal delta-oscillations by MK-801 in Grin1ΔPV mice. (a) Example local field potential (LFP) traces of 2-s duration recorded in mPFC of control (left, blue) and knockout (right, red) mice before (top) and 27 min after (bottom) injection of 0.2 mg kg−1 MK-801. (b) Power spectra calculated for LFP recordings of 10 min, either immediately before (light blue/red) or 20–30 min after (dark blue/red) injection of MK-801 and normalized to total power spectral density. Gray bar highlights the area of 3–5 Hz containing the peak of the induced delta-oscillations and used for the analysis in c. (c) Power in the 3–5 Hz range normalized to total power spectral density and expressed as a ratio of 20–30 min to −10–0 min (baseline). Note that delta power generally increases over time in all conditions (ratio>1) including after vehicle injection (light blue/red, within-subject comparison), but only increases dramatically in MK-801-injected knockouts (dark red), not MK-801-injected controls. Error bars represent s.e.m., asterisk represents significant difference (see main text), n=3 per group.

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