Building upon our previous work this year we have focused on the following four research questions. (1) Are there any other phenotypes which are reminiscent of schizophrenia in Ppp1r2-Cre/NR1 mutants? In human schizophrenia, impaired gamma oscillations are consistently reported and are thought to play a crucial role in a number of the symptoms. We already know that synchronized firing of cortical principal neurons is disturbed in the mutant NR1 mice, which may result in the abnormal oscillatory activity in the cortex. To directly investigate gamma oscillations in mutant mice, we first conducted local field potential (LFP) recoding in vivo before and after injection of a psychostimulant, Modafinil, which is known to induce robust and persistent gamma band oscillatory activity in mouse cortex. We found that, while the magnitude at the gamma frequency in floxed-NR1 control mice was enhanced 30-60 min after Modafinil injection, there was no such change in the mutant mice. To measure more physiologically-relevant gamma oscillatory activity, using recorded auditory-evoked gamma oscillations, we recorded spontaneous LFP activity and tone evoked potential from the auditory cortex of awake mice placed in a stereotaxic apparatus. Forty Hz-click trains (80-dB)-evoked gamma oscillation, normalized to the tone-evoked response, was diminished in the mutant mice. On the other hand, the magnitude of spontaneous LFP fluctuations from mutant auditory cortex was enhanced. This suggests that GABAergic interneuron specific NMDAR hypofunction results in noisy spontaneous LFP fluctuations, which may impair synchronous gamma activity during sensory processing. Another major hallmark of human schizophrenia is an elevated subcortical dopamine release during psychosis. We explored the possibility of a similar dysregulation of dopaminergic tones in the Ppp1r2-Cre/NR1 KO mutants. First, we assessed the psychostimulant-induced hyperlocomotion, which is supposed to be caused by increase in dopamine release in the striatum. We found that amphetamine (Amph)-evoked locomotor activity of the mutants is much higher than control littermates, suggesting an enhanced dopaminergic tone in the striatum in response to psychostimulant, which is seen in schizophrenia patients. Indeed, we confirmed an elevated dopamine release upon Amph injection by microdialysis, in collaboration with Daniel Paredes at NINDS. These two results, impaired gamma oscillation and dopamine dysregulation, provide additional schizophrenia-related pathophysiology to Ppp1r2-Cre/NR1 KO mutants. (2) How does social stress exacerbate the behavioral phenotypes of Ppp1r2-Cre/NR1 KO mutants? A growing body of evidence suggests that redox dysfunction and oxidative stress in brains during neurodevelopment may play a significant role in the etiology of schizophrenia. Previously we also demonstrated that social stress precipitates a number of the pathological phenotypes in the Ppp1r2-Cre/NR1 mutants. This environmental interaction has been hypothesized to involve alteration in oxidative stress mechanisms. To test this hypothesis, we investigated whether oxidative stress is involved in the emergence of schizophrenia-like behaviors in this animal model. Consistent with this idea, post-weaning social isolation (PWSI) augmented oxidative stress levels particularly in cortical PV interneurons and precipitated anxiety-like behavior in 8 week old mutants, a point at which group-housed animals displayed no such behavior. Interestingly, in mPFC of adult mutants we found decreased expression (both mRNA and protein) of a master regulator of mitochondria energy metabolism and anti-oxidation, PGC1 (peroxisome proliferator activated receptor gamma coactivator 1), which is normally highly expressed in fast-spiking PV interneurons. PWSI exacerbated the down-regulation of PGC1 in the mutants. Therefore, elevation of oxidative stress level in mutants appears to be due to impaired anti-oxidant defenses system, which occurs as a downstream consequence of the depletion of NMDARs in early postnatal corticolimbic interneurons. Chronic treatment with the antioxidant/NOX inhibitor apocynin, beginning from postnatal 2 weeks, largely diminished the signs of oxidative stress and alleviated anxiety and the spatial working memory deficit. Our results suggest that oxidative stress plays a critical role in PV interneuron dysfunction presumably through PGC1 downregulation and in the pathogenesis of schizophrenia-like phenotypes. (3) Is NMDAR hypofunction in interneurons really crucial for schizophrenia-related phenotypes? It is unclear whether NR1 ablation at other cell types, such as excitatory neurons, could also induce similar effects. To advance our investigation of whether NMDAR hypofunction-induced schizophrenia-like phenotypes may stem from interneuronal dysfunction, we utilized the Cre/loxP system to generate transgenic conditional knockout mice (G35-3-Cre/fNR1 KO), in which NR1 deletion is largely confined to cortical glutamatergic neurons. In contrast with interneuron-specific NR1 KO mice, we found that these mice do not exhibit comparable significant schizophrenia-like phenotypes in tests for working memory, social behavior, psychostimulant sensitivity, and anhedonia or amotivation. Only prepulse inhibition (PPI) of acoustic startle was mildly impaired in the mutants. This supports the refined hypothesis that NMDAR hypofunction must occur in cortical interneurons in order to model the hallmark symptoms of schizophrenia in rodents. (4) What is the impact of GABA decrease or Gad67 reduction on physiology and behavior? We previously observed Cre-targeted neuron-specific decrease in Gad67 levels in the Ppp1r2-Cre/NR1 mutants. Naturally, the next logical question was which, if any, of the phenotypes seen in the NR1 mutants can be explained by the decrease in Gad67. Alternatively, Gad67 reduction and further GABA level decrease have also been consistently reported in major depressive disorders (MDD) patients. Therefore, to directly assess the impact of Gad67 reduction in psychiatric disorder phenotypes, using the same Ppp1r2-Cre line we generated and characterized a novel transgenic mouse line in which Gad67, was ablated following postnatal day 7 selectively in 50% of cortical and hippocampal interneurons. Gad67 expression was reduced by 50% with a concomitant increase in Gad65 and the GABA levels in the mutant cortex and hippocampus were also significantly reduced after adolescence. Consistent with the comorbidity of epilepsy with depression, subclinical epileptiform discharges were observed by week 11. Furthermore, this manipulation recapitulated several specific features common to major depression including anhedonia, a lack of motivated behavior, and pronounced social withdrawal. However, there were no impairments in tests of behavioral despair or schizophrenia-related behaviors (prepulse inhibition and spatial working memory). Perhaps consistent with this specific behavioral phenotype, in the nucleus accumbens baseline dopamine was elevated while amphetamine-evoked dopamine release was severely attenuated and the hyper-locomotor behavioral response to amphetamine was reduced in the mutants. In all, these results suggest a relationship between the anhedonia aspect of major depressive disorders and impairments in subcortical dopamine regulation through alterations in cortical interneuron networks. In sum, we can now suggest that the schizophrenia-like symptoms caused by disruptions in NMDA signaling may be uniquely due to cortical and hippocampal interneurons. Furthermore, it is becoming increasingly clear that alterations in oxidative stress may be crucial to the development of these phenotypes and that disruptions in gamma oscillations may play a fundamental role in the expression of the pathological condition.
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