The NMDA receptor GluN2C subunit controls cortical excitatoryinhibitory balance, neuronal oscillations and cognitive function

Despite strong evidence for NMDA receptor (NMDAR) hypofunction as an underlying factor for cognitive disorders, the precise roles of various NMDAR subtypes remains unknown. The GluN2Ccontaining NMDARs exhibit unique biophysical properties and expression pattern, and lower expression of GluN2C subunit has been reported in postmortem brains from schizophrenia patients. We found that loss of GluN2C subunit leads to a shift in cortical excitatory-inhibitory balance towards greater inhibition. Specifically, pyramidal neurons in the medial prefrontal cortex (mPFC) of GluN2C knockout mice have reduced mEPSC frequency and dendritic spine density and a contrasting higher frequency of mIPSCs. In addition a greater number of perisomatic GAD67 puncta was observed suggesting a potential increase in parvalbumin interneuron inputs. At a network level the GluN2C knockout mice were found to have a more robust increase in power of oscillations in response to NMDAR blocker MK- 801. Furthermore, GluN2C heterozygous and knockout mice exhibited abnormalities in cognition and sensorimotor gating. Our results demonstrate that loss of GluN2C subunit leads to cortical excitatoryinhibitory imbalance and abnormal neuronal oscillations associated with neurodevelopmental disorders

Deletion of Glutamate Delta-1 Receptor in Mouse Leads to Aberrant Emotional and Social Behaviors

The delta family of ionotropic glutamate receptors consists of glutamate δ1 (GluD1) and glutamate δ2 (GluD2) receptors. While the role of GluD2 in the regulation of cerebellar physiology is well understood, the function of GluD1 in the central nervous system remains elusive. We demonstrate for the first time that deletion of GluD1 leads to abnormal emotional and social behaviors. We found that GluD1 knockout mice (GluD1 KO) were hyperactive, manifested lower anxiety-like behavior, depression-like behavior in a forced swim test and robust aggression in the resident-intruder test. Chronic lithium rescued the depression-like behavior in GluD1 KO. GluD1 KO mice also manifested deficits in social interaction. In the sociability test, GluD1 KO mice spent more time interacting with an inanimate object compared to a conspecific mouse. D-Cycloserine (DCS) administration was able to rescue social interaction deficits observed in GluD1 KO mice. At a molecular level synaptoneurosome preparations revealed lower GluA1 and GluA2 subunit expression in the prefrontal cortex and higher GluA1, GluK2 and PSD95 expression in the amygdala of GluD1 KO. Moreover, DCS normalized the lower GluA1 expression in prefrontal cortex of GluD1 KO. We propose that deletion of GluD1 leads to aberrant circuitry in prefrontal cortex and amygdala owing to its potential role in presynaptic differentiation and synapse formation. Furthermore, these findings are in agreement with the human genetic studies suggesting a strong association of GRID1 gene with several neuropsychiatric disorders including schizophrenia, bipolar disorder, autism spectrum disorders and major depressive disorder

Deletion of glutamate delta-1 receptor in mouse leads to enhanced working memory and deficit in fear conditioning.

Glutamate delta-1 (GluD1) receptors are expressed throughout the forebrain during development with high levels in the hippocampus during adulthood. We have recently shown that deletion of GluD1 receptor results in aberrant emotional and social behaviors such as hyperaggression and depression-like behaviors and social interaction deficits. Additionally, abnormal expression of synaptic proteins was observed in amygdala and prefrontal cortex of GluD1 knockout mice (GluD1 KO). However the role of GluD1 in learning and memory paradigms remains unknown. In the present study we evaluated GluD1 KO in learning and memory tests. In the eight-arm radial maze GluD1 KO mice committed fewer working memory errors compared to wildtype mice but had normal reference memory. Enhanced working memory in GluD1 KO was also evident by greater percent alternation in the spontaneous Y-maze test. No difference was observed in object recognition memory in the GluD1 KO mice. In the Morris water maze test GluD1 KO mice showed no difference in acquisition but had longer latency to find the platform in the reversal learning task. GluD1 KO mice showed a deficit in contextual and cue fear conditioning but had normal latent inhibition. The deficit in contextual fear conditioning was reversed by D-Cycloserine (DCS) treatment. GluD1 KO mice were also found to be more sensitive to foot-shock compared to wildtype. We further studied molecular changes in the hippocampus, where we found lower levels of GluA1, GluA2 and GluK2 subunits while a contrasting higher level of GluN2B in GluD1 KO. Additionally, we found higher postsynaptic density protein 95 (PSD95) and lower glutamate decarboxylase 67 (GAD67) expression in GluD1 KO. We propose that GluD1 is crucial for normal functioning of synapses and absence of GluD1 leads to specific abnormalities in learning and memory. These findings provide novel insights into the role of GluD1 receptors in the central nervous system

Lithium rescues depression-like behavior in GluD1 KO.

<p><b>A.</b> Open field test was performed on four groups, control diet WT (n = 7), lithium WT (n = 9), control diet GluD1 KO (n = 8) and lithium GluD1 KO (n = 7). In the open field test no drug X genotype effect was observed in the number of line crossings (two-way ANOVA, drug F(1, 27) = 0.8275, P = 0.3710; genotype F(1, 27) = 5.678, P = 0.0245; interaction F(1, 27) = 2.589, P = 0.1192). <b>B.</b> Forced swim test was performed on control diet WT (n = 12), lithium WT (n = 13), control diet GluD1 KO (n = 11) and lithium GluD1 KO (n = 11). Lithium reduced the immobility time in forced swim test in the GluD1 KO (two-way ANOVA, drug F(1, 43) = 9.717, P = 0.0033; genotype F(1, 43) = 80.49, P<0.0001; interaction F(1, 43) = 15.84, P = 0.0003). <b>C.</b> Resident-intruder test was performed on control diet WT (n = 5), lithium WT (n = 5), control diet GluD1 KO (n = 6) and lithium GluD1 KO (n = 5). Lithium failed to rescue the higher attack frequency in GluD1 KO in the resident-intruder test (attack frequency: two-way ANOVA, drug F(1, 17) = 0.3570, P = 0.5581; genotype F(1, 17) = 17.12, P = 0.0007; interaction F(1, 17) = 0.9182, P = 0.3514). Data are presented as mean ± SEM. *** represents P<0.001.</p

GluD1 KO are hyperaggressive.

<p><b>A.</b> In the resident-intruder test for aggression GluD1 KO mice (n = 12) manifested robust aggression with a higher attack frequency compared to WT mice (n = 9) (attack frequency, unpaired t-test with Welch's correction, P = 0.0003, F = 6.577). <b>B.</b> GluD1 KO mice (n = 12) have a significantly shorter attack latency compared to WT mice (n = 9) (unpaired t-test, P = 0.0105, F = 4.605). Data are presented as mean ± SEM. *** represents P<0.001 and * represents P<0.05.</p

Altered expression of iGluR subunits and synaptic proteins in GluD1 KO.

<p><b>A.</b> RT-PCR showed expression of the GluD1 mRNA in the amygdala, hippocampus and the prefrontal cortex of P15 WT mice. No expression of GluD1 mRNA was seen in GluD1 KO. <b>B.</b> Synaptoneurosomes were collected (5–11 animals each for WT and GluD1 KO) and western blotting analysis was performed for iGluR subunits and synaptic proteins. In synaptoneurosomal preparations from the amygdala of GluD1 KO and WT mice we found a significantly higher expression of GluA1 (P = 0.0486), GluK2 (P = 0.0327) and PSD95 (P = 0.0028) and a trend for higher expression of GAD67 (P = 0.0640) in GluD1 KO. In the prefrontal cortex we observed a significantly lower expression of GluA1 (P<0.001) and GluA2 (P = 0.0345) in GluD1 KO. Data are presented as mean ± SEM. *** represents P<0.001, ** represents P<0.01 and * represents P<0.05.</p

Synaptic protein composition in GluD1 KO mice.

<p>Synaptoneurosomes were isolated from the GluD1 KO and WT amygdala and prefrontal cortex and western blotting was performed (5–11 animals for each group). Data are presented as mean ± SEM. First, the optical density of each sample was normalized to β-actin. Thereafter, the optical density was normalized to the mean of the WT samples. The average ± SEM of optical densities of GluD1 KO samples, that were normalized to WT mean, are represented as Ratio (KO/WT) ± SEM. The P values were calculated from optical densities of WT and GluD1 KO samples normalized to the WT mean.</p>***<p>represents P<0.001.</p>**<p>represents P<0.01 and.</p>*<p>represents P<0.05.</p

DCS rescues social interaction deficits in GluD1 KO.

<p><b>A.</b> WT and GluD1 KO mice were intraperitoneally injected with DCS (320 mg/kg) or saline 20 min prior to sociability test (n = 6 for each group). DCS administration in GluD1 KO significantly enhanced percent time spent interacting with Stranger 1 compared to the empty container (two-way ANOVA, drug F(1, 20) = 34.28, P<0.0001; genotype F(1, 20) = 2.103, P = 0.1625; interaction F(1, 20) = 44.79, P<0.0001). <b>B.</b> In the test of social novelty GluD1 KO showed significantly higher percent time interacting with Stranger 2 compared to Stranger 1 (two-way ANOVA, drug F(1, 20) = 3.897, P = 0.0624; genotype F(1, 20) = 5.811, P = 0.0257; interaction F(1, 20) = 29.66, P<0.0001). Data are presented as mean ± SEM. *** represents P<0.001.</p

DCS normalized the reduced GluA1 expression in prefrontal cortex in GluD1 KO.

<p>Synaptoneurosomes were prepared from amygdala and prefrontal cortex 2 hours after the end of the social novelty test in which saline or DCS (320 mg/kg) was administered intraperitoneally (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032969#pone-0032969-g007" target="_blank">Fig. 7</a>) (n = 3 for each group). GluA1 expression was higher in GluD1 KO amygdala and lower in the prefrontal cortex. DCS did not normalize the higher GluA1 expression in amygdala (two-way ANOVA, drug F(1,8) = 0.7175, P = 0.4216; genotype F(1,8) = 35.59, P = 0.0003; interaction F(1,8) = 0.2622, P = 0.6224). In the prefrontal cortex the lower GluA1 expression was increased to the WT levels by DCS treatment (two-way ANOVA, drug F(1,8) = 11.57, P = 0.0093; genotype F(1,8) = 5.884, P = 0.0415; interaction F(1,8) = 6.140, P = 0.0382). Data are presented as mean ± SEM. * represents P<0.05.</p

GluD1 KO exhibit depression-like behavior.

<p><b>A.</b> In a forced swim test GluD1 KO (n = 13) displayed a greater duration of immobility compared to WT mice (n = 11) (unpaired t-test, P<0.0001, F = 1.060). <b>B.</b> GluD1 KO (n = 13) had a significantly shorter latency to immobility compared to WT mice (n = 11) (unpaired t-test, P = 0.0040, F = 3.446). <b>C.</b> GluD1 KO mice (n = 5) manifested significantly lower preference for sucrose compared to WT (n = 7) mice (unpaired t-test, P = 0.0118, F = 2.275). Data are presented as mean ± SEM. *** represents P<0.001 and ** represents P<0.01 and * represents P<0.05.</p