Postnatal nmda receptor ablation in corticolimbic interneurons confers schizophrenia-like phenotypes anoxia meaning in hindi

Cortical gabaergic dysfunction may underlie the pathophysiology of psychiatric disorders, including schizophrenia. Here, we characterized a mouse strain in which the essential NR1 subunit of the NMDA receptor (NMDAR) was selectively eliminated in 40–50% of cortical and hippocampal interneurons in early postnatal development. Consistent with the NMDAR hypofunction theory of schizophrenia, distinct schizophrenia-related symptoms emerged after adolescence, including novelty-induced hyperlocomotion, mating and nest-building deficits, as well as anhedonia-like and anxiety-like behaviors. Many of these behaviors were exacerbated by social isolation stress.

Social memory, spatial working memory and prepulse inhibition were also impaired.Anoxia meaning in hindi reduced expression of glutamic acid decarboxylase 67 and parvalbumin was accompanied by disinhibition of cortical excitatory neurons and reduced neuronal synchrony. Postadolescent deletion of NR1 did not result in such abnormalities. These findings suggest that early postnatal inhibition of NMDAR activity in corticolimbic gabaergic interneurons contributes to the pathophysiology of schizophrenia-related disorders.

Postmortem brains of schizophrenia subjects suggest that dysfunction of gabaergic interneurons, particularly those containing the calcium-binding protein parvalbumin, may be a core feature of schizophrenia 10, 11. Supporting this notion, reduced expression of the GABA-synthesizing enzyme glutamic acid decarboxylase 67 (GAD67) 12 and parvalbumin 13 have been found in cortical interneurons of individuals with schizophrenia.Anoxia meaning in hindi the possibility that corticolimbic gabaergic interneurons are a prime target for NMDAR hypofunction 14, 15 is supported by three lines of evidence. First, acute systemic administration of NMDAR antagonists results in hyperactivity of cortical pyramidal neurons 16 and spillover of cortical acetylcholine 17 and glutamate 18. These paradoxical cellular changes concur with brain-imaging data showing net cortical excitation after NMDA antagonist treatment in human subjects 19, 20. Second, gabaergic interneurons are disproportionally more sensitive to NMDAR antagonists than pyramidal neurons 15, 21, although the precise mechanisms for this difference is unresolved. NMDAR antagonist–induced cortical excitation may be a result of a preferential reduction in the firing of fast-spiking interneurons and resultant disinhibition of cortical excitatory neurons.Anoxia meaning in hindi third, repeated administration of NMDAR antagonists decreases GAD67 and parvalbumin expression in cortical gabaergic neurons 22– 26, linking NMDAR hypofunction to dysfunction of gabaergic neurons.

We generated a cre recombinase transgenic line in which cre expression was driven by the ppp1r2 gene promoter (protein phosphatase 1, regulatory (inhibitor) subunit 2) ( fig. 1). Endogenous ppp1r2 is expressed in gabaergic neurons in the striatum, cortex, hippocampus and, to a lesser degree, pyramidal neurons in hippocampal CA1 ( fig. 1a and supplementary fig. 1). When crossed with a loxp-flanked rosa26-lacz reporter mouse, our new line cre #4127 ( ppp1r2-cre +/−; referred to here as ppp1r2-cre or simply cre) resulted in a scattered distribution of lacz-positive neurons throughout the prefrontal cortex, neocortex and hippocampus ( fig. 1c–e and supplementary fig. 1).Anoxia meaning in hindi the dependence of cre recombinase expression on the ppp1r2 promoter was confirmed by coexpression of lacz and ppp1r2 ( fig. 1b). Over 92% of lacz-positive neurons were gabaergic, as demonstrated by their coexpression of GAD67 ( fig. 1f and supplementary fig. 2), and consisted of 40–50% of the cortical gabaergic neurons across several cortical regions. No lacz-positive neurons colocalized with the excitatory neuron markers TBR1 ( fig. 1g) or αcamkii ( supplementary fig. 2), suggesting that cre recombination occurred exclusively in gabaergic neurons.

Cre recombination was detected in the cortex and hippocampus first at postnatal day 7 and the restriction of lacz expression to gabaergic neurons was maintained for up to 18 weeks ( supplementary fig. 3).Anoxia meaning in hindi immunofluorescence staining in primary somatosensory cortex (S1) at 8 weeks revealed that 70% of the lacz-positive neurons were parvalbumin positive ( fig. 1h) and 24% of the remaining population was labeled by antibody to reelin ( supplementary fig. 2). In complementary analysis, cre-targeted neurons represented 75% of parvalbumin-positive interneurons and 30% of the reelin-positive interneurons. In contrast, less than 3% of cre-targeted neurons in the cortex were colabeled with antibody to somatostatin ( supplementary fig. 2) and 15% of cre-targeted neurons were labeled by antibody to neuropeptide Y ( supplementary fig. 2). No colocalization was observed with antibody to calretinin in any brain area ( fig. 1i).Anoxia meaning in hindi thus cre-loxp recombination in the ppp1r2-cre line occurred early in postnatal development in 40–50% of cortical and hippocampal gabaergic interneurons, the majority of which were parvalbumin positive.

We crossed the ppp1r2-cre mice with the loxp-flanked NR1 line A ( NR1 loxp/loxp-line A or simply flox-A) 27 to restrict NMDAR ablation to cre-targeted gabaergic neurons. Homozygous NR1 loxp/loxp; ppp1r2-cre progeny ( ppp1r2-cre +/−; NR1 loxp/loxp-line A, hereafter postnatal knockout mutant or simply mutant) were viable and fertile, with no gross morphological abnormalities revealed by nissl staining. The spatial and temporal pattern of NR1 mrna was analyzed by in situ hybridization ( fig. 2a–c).Anoxia meaning in hindi expression of NR1 mrna in control mice (cre and flox-A) was detected in most neurons expressing gad67 mrna. In contrast, NR1 mrna expression was absent in 40–50% of cortical and hippocampal GAD67-positive neurons in 4-week-old mutant mice ( fig. 2c), suggesting that NR1 was knocked out in cre-targeted gabaergic neurons. This pattern was maintained in the mutant mice up to 20 weeks, with no noticable decrease in NR1 mrna being detected in other areas, including striatum, basolateral amygdala, thalamic reticular nucleus, cerebellum and the nucleus tractus solitarius ( fig. 2c and supplementary fig. 4). No difference was found for the ratio of gad67 mrna–positive neurons to the total number of neurons between genotypes, demonstrating an absence of neurodegeneration following NR1 ablation (medial prefrontal cortex, 20.1 ± 2.3% for control ( ppp1r2-cre and flox-A, n = 4 in total) versus 19.6 ± 3.4% for mutant ( n = 3), t test, P = 0.91; S1 cortex, 21.5 ± 2.3% for control versus 20.3 ± 3.5% for mutant, t test, P = 0.76).Anoxia meaning in hindi moreover, there were no differences among genotypes in the number of NR1 mrna–positive, gad67 mrna–negative neurons in the cre-targeted areas, including the CA1 pyramidal neuron layer of the hippocampus at 20 weeks of age, indicating that there was no reduction of NR1 mrna in excitatory neurons ( supplementary fig. 5).

To evaluate whether NMDAR function was absent in the cre-targeted neurons, we induced coexpression of enhanced yellow fluorescent protein (EYFP) by cre recombination ( fig. 2d) by crossing mutants or pppr12-cre mice with a loxp-flanked rosa26-EYFP line. Whole-cell patch-clamp recordings of EYFP-expressing neurons in S1 and hippocampal CA1 were performed in mutants ( ppp1r2-cre +/−; NR1 loxp/loxp-line A; rosa26-EYFP loxp/+) and cre controls ( ppp1r2-cre +/−; rosa26-EYFP loxp/+) at 6–26 weeks of age.Anoxia meaning in hindi in the presence of the AMPA-type glutamate receptor channel blocker 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX), spontaneous excitatory postsynaptic currents (sepsc) were observed in all the neurons examined in cre-control mice, including fast-spiking neurons ( fig. 2e,f and supplementary fig. 6). SEPSCs were mediated by NMDAR channels, as they were subsequently blocked by the addition of the NMDA channel blocker d(−)-2-amino-5-phosphonovaleric acid ( d-AP5). In contrast, in the presence of NBQX, no sepscs were observed in the S1 of the mutant mice and no further blockade was observed following the addition of d-AP5. EYFP-positive neurons in the hippocampal CA1 subfields of mutant mice yielded similar results ( supplementary fig. 6).Anoxia meaning in hindi kinetic analysis under baseline conditions also showed that the mean decay time of sepscs in cre-targeted neurons in the mutant mice was significantly shorter than in cre-controls (mann-whitney U test, P 0.01, supplementary fig. 6), suggesting the absence of the slow NMDAR component of the sepsc in the mutants. Immunocytochemical analysis confirmed that all of the biocytin filled–neurons during whole-cell patch-clamp recording of EYFP-positive neurons colocalized with GAD67. Many neurons also colocalized with parvalbumin ( fig. 2d) and some displayed fast-spiking, nonadapting firing patterns ( supplementary fig. 6). Together, this suggests that NMDAR was functionally eliminated from the cre-targeted gabaergic neurons in the mutant mice.Anoxia meaning in hindi

To assess the behavioral consequence of knocking out NMDAR in corticolimbic gabaergic neurons, we subjected mutant ( ppp1r2-cre +/−; NR1 loxp/loxp-line A) males and age-matched control (flox-A line, NR1 loxp/loxp-line A or cre, ppp1r2-cre +/−) males (8–18 weeks old) to a battery of behavioral tests. Mice were single-housed for 1 week before testing unless otherwise noted. The mutant mice had no significant impairment in basic functions, including body growth rate, thermoregulation, basic auditory, visual and olfactory function, pain sensitivity and motor coordination ( supplementary fig. 7). Spatial acuity, assessed by visually evoked potentials, was also normal. No obvious locomotor abnormality was apparent at 9–10 weeks of age ( supplementary fig. 8).Anoxia meaning in hindi however, novelty-induced hyperlocomotion was prominent in the mutant mice during the first 3 min of spontaneous exploration in an open-field test ( fig. 3a), and they also spent substantially less time than controls exploring the unprotected center area of the open field ( fig. 3b), consistent with an anxiety-like phenotype. To explicitly test for anxiety-like behavior in the mutant mice, we subjected naive mice to an elevated plus-maze task. Group-housed mutant mice (3–5 per cage) exhibited clear anxiety-like behavior at 16 weeks of age, but not at 8 weeks ( fig.3c and supplementary fig. 8). This phenotype was precipitated by social isolation–induced stress and became evident in 8-week-old mutants that were single-housed for a week ( fig. 3c).Anoxia meaning in hindi

To extend our analysis of the mutant to social behaviors, we subjected 12-week-old mutant mice and control littermates to a social-recognition test in a novel cage ( fig. 3e). Control mice demonstrated social investigation (intense sniffing) toward a 4-week-old ‘stimulus male’ during the first 1-min presentation but spent less time investigating the same mouse in subsequent presentations, reflecting an intact social short-term memory. During the last trial, subjects were presented with a new stimulus male and controls again demonstrated social investigation (increased sniffing), suggesting normal social recognition and intact social short-term memory. In contrast, the mutant mice did not investigate the stimulus mouse during the first 1-min presentation, which may be a result of enhanced novelty-induced anxiety ( supplementary fig. 9).Anoxia meaning in hindi however, mutant mice showed increased social investigation toward the stimulus mouse after the second trial, which was maintained in subsequent trials without habituation ( fig. 3e). If the stimulus male was present continuously for 10 min in a subsequent test, both the mutant and control mice displayed decreased social investigation ( supplementary fig. 9). This suggests that the impaired habituation of social investigation by the mutants may be a result of a deficit in short-term memory. To test for a short-term memory deficit, we conducted a test of spontaneous Y-maze alternations, a spatial working memory task based on the natural tendency of mice to alternate the choice of maze arms. Although reliable alternation was observed in control mice, mutant mice displayed a reduction in alternation to chance levels ( fig. 3f), suggesting the presence of a spatial working memory deficit, which is well documented in schizophrenia 28.Anoxia meaning in hindi

Behavioral analysis of single-housed adult knockout mutant males and flox-B control littermates were conducted 5 weeks after the completion of NR1 knockout (at 25 weeks) to parallel the time course used in the postnatal knockout experiments. Adult knockout mutants did not show the behavioral deficits observed in the postnatal knockout mutant mice and displayed normal social recognition ( fig. 6b), normal PPI of the auditory startle reflex ( fig. 6c) and normal behavior in the elevated plus maze (4.87 ± 1.2 entries to open arms for flox-B control versus 3.82 ± 1.4 for adult knockout mutant, t test, P = 0.58) and in the Y-maze spontaneous alternation task (alternation index (%); 65.8 ± 6.1 for flox-B control versus 65.2 ± 4.6 for adult knockout mutant, t test, P = 0.93).Anoxia meaning in hindi even at 32 weeks of age, adult knockout mutants did not have a nest-building impairment (0.02 ± 0.01 g of unused nestlet for flox-B control versus 0.016 ± 0.02 g for adult knockout mutant) or anxiety-like behaviors (data not shown), indicating that the absence of deficits in adult knockout mutants was not a result of a delayed onset of schizophrenia-related symptoms. There was also no reduction in GAD67 or parvalbumin immunoreactivity in the cre-targeted neurons of the adult knockout mutants immediately after the completion of NR1 deletion (20 weeks old; data not shown) or at 26–27 weeks ( fig. 6d), suggesting that gabaergic dysfunction in adult knockout mutants was negligible. Moreover, in vivo electrophysiological recordings revealed no differences in mean firing rates or cross-correlation values between nearby pyramidal neurons in adult knockout mutants versus controls ( fig. 7a–c).Anoxia meaning in hindi collectively, these results suggest that early postnatal NMDAR hypofunction in corticolimbic gabaergic neurons is crucial for the development of schizophrenia-related symptoms in mice.

Our postnatal NR1 knockout mutant mice share several pathophysiological features that are typical of major psychiatric disorders, especially, the constellation of symptoms found in schizophrenia. In particular, the mutant mice exhibited both positive symptoms, such as psychomotor agitation, and negative symptoms, such as a reduced preference for sweet solution and deficits in nesting/mating, which mirror anhedonia and social withdrawal. In addition, the mutant mice had cognitive symptoms such as deficits in spatial working memory and short-term social memory.Anoxia meaning in hindi impaired sensorimotor gating, as indicated by decreased PPI of the startle reflex in our mutant mice, is also often observed in individuals with schizophrenia 29. Similarly, the NR1-deleted cortical gabaergic neurons had reduced GAD67 and parvalbumin levels, concurring with reduced expression of these markers 12, 13 in the postmortem cortex of individuals with schizophrenia. The disinhibition of cortical excitatory neurons and reduced neuronal synchrony that we observed in the postnatal knockout mutants is also consistent with hyperactivity of the dorsolateral prefrontal cortex during a working-memory task, which is seen in individuals with schizophrenia 32.

We thank N. Heintz for pld53.SCAEB plasmid and the BAC homologous recombination protocol, F.Anoxia meaning in hindi costantini for the loxp-flanked rosa26-EYFP mouse strain, D.L. Brautigan for antibody to ppp1r2, B. Condie and J. Rubenstein for gad67 cdna, J. Pickel for oocyte injections, J.N. Crawley for advice on behavioral testing, S. Zhang, J. Okolonta and M. Taylor for technical and animal care assistance, H. Matsunami for in situ hybridization protocol, Y. Kubota for immunostaining protocol and J. Yamamoto for neuralynx/xclust2 conversion software. We thank D.R. Weinberger, M.M. Behrens, G. Kunos, I. Henter, K.M. Christian, H. Giesen and H.A. Nash for critical comments on the manuscript. We also acknowledge the CURE/digestive diseases research center at the university of california los angeles and the US national institute of mental health chemical synthesis and drug supply program for antibodies and risperidone, respectively.Anoxia meaning in hindi this work was supported by the intramural research program of the US national institute of mental health and of the US national institute on alcohol abuse and addiction.