Characteristics of sEPSCs in mPFC layer II/III pyramidal neurons.

*<p><i>p</i><0.02 between genotypes.</p>**<p><i>p</i><0.01 between genotypes. Data are mean ± s.e.m.</p

Normal MK-801-induced hyperlocomotion.

<p>No difference for genotype (black for mutant (n = 8), white for Flox mice (n = 9)) in MK-801-induced locomotor response (two-way repeated measures ANOVA after treatment; <i>p</i> = 0.72).</p

Contribution of NMDA Receptor Hypofunction in Prefrontal and Cortical Excitatory Neurons to Schizophrenia-Like Phenotypes

<div><p>Pharmacological and genetic studies support a role for NMDA receptor (NMDAR) hypofunction in the etiology of schizophrenia. We have previously demonstrated that NMDAR obligatory subunit 1 (GluN1) deletion in corticolimbic interneurons during early postnatal development is sufficient to confer schizophrenia-like phenotypes in mice. However, the consequence of NMDAR hypofunction in cortical excitatory neurons is not well delineated. Here, we characterize a conditional knockout mouse strain (CtxGluN1 KO mice), in which postnatal GluN1 deletion is largely confined to the excitatory neurons in layer II/III of the medial prefrontal cortex and sensory cortices, as evidenced by the lack of GluN1 mRNA expression in <i>in situ</i> hybridization immunocytochemistry as well as the lack of NMDA currents with <i>in vitro</i> recordings. Mutants were impaired in prepulse inhibition of the auditory startle reflex as well as object-based short-term memory. However, they did not exhibit impairments in additional hallmarks of schizophrenia-like phenotypes (<i>e.g.</i> spatial working memory, social behavior, saccharine preference, novelty and amphetamine-induced hyperlocomotion, and anxiety-related behavior). Furthermore, upon administration of the NMDA receptor antagonist, MK-801, there were no differences in locomotor activity versus controls. The mutant mice also showed negligible levels of reactive oxygen species production following chronic social isolation, and recording of miniature-EPSC/IPSCs from layer II/III excitatory neurons in medial prefrontal cortex suggested no alteration in GABAergic activity. All together, the mutant mice displayed cognitive deficits in the absence of additional behavioral or cellular phenotypes reflecting schizophrenia pathophysiology. Thus, NMDAR hypofunction in prefrontal and cortical excitatory neurons may recapitulate only a cognitive aspect of human schizophrenia symptoms.</p></div

Integrity of hippocampus and amygdala.

<p><b>A</b> and <b>B.</b> Eleven-week-old G35-3-Cre/R26lacZ mice showed robust Cre recombination (blue signals, left panel) in both hippocampal pyramidal neurons (A) and amygdalal principal neurons in lateral and basolateral amygdala nuclei (B). However, <i>in situ</i> hybridization revealed no clear reduction of GluN1 mRNA levels was detected in both brain regions of the mutant mice compared to Flox controls (right two panels) at the same age. In situ hybridization images corresponded to the white box area in the left panel. Scale Bar: 200 µm. <b>C.</b> In the spatial long-term memory task, both fGluN1 (while bar, n = 7) and mutant (black bar, n = 8) mice preferentially visited the novel arm during the test trial (two-way ANOVA/Fisher’s LSD <i>post-hoc</i> test: *<i>p</i><0.05). <b>D.</b> In Y-maze spontaneous alternation task, no difference between fGluN1 (Flox, n = 17) and mutant (n = 16) mice in alteration percentage or arm entries. <b>E.</b> In the contextual fear conditioning test, there was no difference between the fGluN1 control (white bar, n = 9) and mutant (black bar, n = 10) mice for freezing in the training (Context A) versus the novel context (Context B) (repeated measures ANOVA; Group, <i>p</i> = 0.99; Group×Context, <i>p</i> = 0.78).</p

Histological characterization of cortical excitatory neuron-selective GluN1 knockout mice.

<p><b>A.</b> Spatial distribution of Cre recombinase activity in coronal sections of G35-3-Cre/R26lacZ mice, stained with X-gal (blue) and Safranin-O. <b>B.</b> High magnification photographs in the medial prefrontal cortex (mPFC). Left, X-gal staining of G35-3-Cre/R26lacZ mice. Right, <i>in situ</i> hybridization images with DIG-labeled GluN1 cRNA (blue) in the mPFC region corresponding to the dotted area in the X-Gal staining image. <b>C</b> and <b>D.</b> High magnification photographs in the primary sensory (S1) cortex. X-Gal staining image in C, and <i>in situ</i> hybridization images with DIG-labeled GluN1 cRNA (blue) in D. Right panels, a to d, are shown from the dotted areas in the left panels from floxed-GluN1 control (Flox) and mutant mice, respectively. All mice were 11 weeks of age. Scale Bar: 200 µm.</p

Reactive oxygen species’ level and GABAergic inhibition.

<p><b>A.</b> Fourteen-week-old mice received injection of dihydroethidium to assess reactive oxygen species (ROS) levels. ROS levels, visualized by red fluorescence, were negligible in cortical sections of somatosensory cortex (upper) and medial prefrontal cortex (lower) in both Flox and CtxGluN1KO mutant mice. Right panels are sections from adult corticolimbic interneuron-GluN1 knockout mice (Ppp1r2-Cre/GluN1 KO mice) as a positive control. Scale Bar: 100 µm. <b>B.</b> Gad67 protein levels in cortical homogenates (6 animals for each group) were examined by Western blot. Gad67-IR intensity was normalized by the β-actin–IR on the same gel. No genotypic difference was detected in Gad67 protein levels. Student’s t-test, <i>p</i> = 0.56. <b>C.</b> Properties of miniature EPSC (mEPSC) and miniature-IPSC (mIPSC) events in mPFC layer II/III pyramidal neurons. The data of 2–3 cells were collected from single animal and averaged per animal (animal number, n = 6 for Flox (white bar), n = 7 for mutant (black bar)). No genotypic differences were observed in frequencies or amplitudes of both measures. Student’s t-test, <i>p</i> = 0.88 for mEPSC frequency, <i>p</i> = 0.97 for mIPSC frequency, <i>p</i> = 0.81 for mEPSC amplitude, <i>p</i> = 0.37 for mIPSC amplitude.</p

Mutants are impaired in prepulse inhibition of the auditory startle reflex.

<p><b>A.</b> Compared with C57BL/6 wild-type (gray bar, n = 14) and fGluN1 (Flox) controls (while bar, n = 21), mutant mice (black bar, n = 20) showed significantly reduced prepulse inhibition of the auditory startle reflex (two-way repeated measures ANOVA; *<i>p</i><0.05, for genotype factor). <b>B.</b> The same mutant mice (black bar) were unimpaired in auditory startle reflex compared to C57BL/6 wild type (gray bar) and Flox (white bar) controls (two-way repeated measures ANOVA; F(2,172) = 0.88, <i>p</i> = 0.42).</p

No NMDA currents detected in most of cortical layer II/III pyramidal neurons and postnatal onset of GluN1 deletion.

<p><b>A.</b> An example trace (right) showing no NMDA currents were detected in mPFC layer II/III pyramidal neurons. Nine cells out of 10 cells tested from 7 mutants (12–17 weeks old) showed no NMDA currents, while they (arrows) were always detected in fGluN1 (Flox) controls. To isolate the NMDA component of spontaneous EPSC events, 20 µM NBQX was bath-applied, and, furthermore, 50 µM D-AP5 was added to ensure that NMDA channel currents were blocked. Scale bar is 500 ms and 50 pA for long traces and 25 ms and 10 pA for average excitatory currents. <b>B.</b> X-gal staining of the primary sensory (S1) cortex on postnatal day 6 (P6) of G35-3-Cre/R26lacZ mice. <b>C.</b> Immunohistochemistry staining for 5-HTT on P6 revealed no difference in barrel field development in the S1 cortex of fGluN1 (Flox) control and mutant mice. Scale bar: 200 µm.</p

Mutants are impaired in multi-object short-term memory task.

<p><b>A.</b> In a standard novel object recognition task with 2-min delay, both Flox controls (white bar, n = 7; two-tailed t-test, *<i>p</i><0.05) and mutant mice (black bar, n = 8; two-tailed t-test, *<i>p</i><0.05) spent more time investigating the novel vs old object. <b>B.</b> Two-min after a five-object exploration trial, Flox controls (while bar, n = 19) spent more time investigating the novel objects during the test trial (two-tailed t-test, **<i>p</i><0.01), while mutant mice (black bar, n = 14) showed no difference for time spent investigating the novel vs old object (two-tailed t-test, <i>p</i> = 0.89). n.s., not significant. <b>C.</b> After a five-object exploration trial with almost no delay (<10 sec), both Flox controls (white bar, n = 8; two-tailed t-test, *<i>p</i><0.05) and mutants mice (black bar, n = 9; two-tailed t-test, *<i>p</i><0.05) spent more time investigating the novel vs old object.</p

Positive and negative-like phenotypes.

<p><b>A.</b> In the novel open field test, no difference in horizontal activity between mutant (black triangle, n = 8) mice and Flox (while square, n = 14) controls (two-way repeated measures ANOVA; <i>p</i> = 0.56). <b>B.</b> No significant difference for genotype (black for mutant (n = 9), white for Flox (n = 9)) in amphetamine-induced locomotor response (two-way repeated measures ANOVA after treatment; <i>p</i> = 0.12). <b>C.</b> No difference between mutant (black, n = 10) and Flox (white, n = 12) mice in the social recognition task (two-way repeated measures ANOVA; <i>p</i> = 0.56). <b>D.</b> In the saccharine preference test, both genotypes consumed saccharine more and no difference between mutant (black, n = 11) and Flox (white, n = 12) mice in saccharine preference (two-way ANOVA/LSD <i>post-hoc</i> test; <i>p</i> = 0.74). <b>E.</b> Mutant mice (black bar, n = 8) performed similarly to Flox controls (while bar, n = 9) in the female urine sniffing test (two-way ANOVA/LSD <i>post-hoc</i> test; <i>p</i> = 0.59). <b>F.</b> Mutant mice (n = 12) were comparable to flox controls (n = 11) in time spent in the open arm of the elevated plus maze (two-tailed t-test; <i>p</i> = 0.73).</p