N-Methyl-D-Aspartate Receptor and Calbindin-Containing Neurons in the Anterior Cingulate Cortex in Schizophrenia and Bipolar Disorder

doi:10.1016/j.biopsych.2008.04.034

Biological Psychiatry

Volume 64, Issue 9, 1 November 2008, Pages 803-809

https://doi.org/10.1016/j.biopsych.2008.04.034 Get rights and content

Background

Glutamatergic modulation of γ-aminobutyric acid (GABA) interneurons via the NR2A subunit of the N-methyl-D-aspartate (NMDA) receptor in the cerebral cortex contributes to the pathophysiology of schizophrenia and bipolar disorder. Previously, we found that, in the anterior cingulate cortex (ACCx), the number of GABA cells that expressed the messenger RNA (mRNA) for the NMDA NR2A subunit was significantly decreased in subjects with schizophrenia and bipolar disorder and that this decrease occurred most prominently in layer 2. In this study, we hypothesized that the subset of GABA interneurons that contained the calcium-binding protein calbindin (CB), by virtue of their preferential localization to layer 2, might be particularly affected.

Methods

We simultaneously labeled the mRNA for the NMDA NR2A subunit with [³⁵S] and the mRNA for CB with digoxigenin with an immunoperoxidase procedure.

Results

We found that, in the normal human ACCx, only approximately 10% of all CB-containing cells expressed NR2A mRNA. However, compared with the normal control subjects and subjects with bipolar disorder, the density of CB+/NR2A+ neurons in layer 2 was increased by 41% to 44 % in subjects with schizophrenia, whereas the amount of NR2A mRNA/CB+ neurons was unchanged.

Conclusions

These observations suggest that, in schizophrenia, a number of CB-containing cells that normally do not express NR2A might become NR2A-expressing or, perhaps not mutually exclusively, the number of CB-expressing cells might be increased and these cells express NR2A. The findings of this study highlight the notion that glutamatergic innervation of subsets of GABA cells might be differentially altered in schizophrenia and bipolar disorder.

Section snippets

Subjects

A cohort of 60 human brains from 20 subjects with schizophrenia, 20 subjects with bipolar disorder, and 20 normal control subjects were obtained from the Harvard Brain Tissue Resource Center at McLean Hospital, Belmont, Massachusetts (Supplement 1). Each group of subjects with schizophrenia or bipolar disorder was matched to a normal control group on the basis of age, postmortem interval (PMI), and whenever possible, gender and side of hemisphere. The mean freezer storage time (days ± SD) of

Statistical Analyses

The densities of CB+ single- labeled neurons were compared between subject groups across layers 2 through 6 with repeated-measures analysis of variance (ANOVA), with diagnosis as the between-groups factor, layer as the within-group factor, and repeated measures on layer. For the CB+/NR2A+ double-labeled neurons, to test the a priori hypothesis that the density of these neurons would be significantly decreased in layer 2 in subjects with schizophrenia and bipolar disorder, a one-way analysis of

Density of All CB+ Neurons

Our repeated-measures ANOVA model failed to reveal any significant diagnosis effect [F(2,57) = 1.09; p = .34] on the density of these neurons (Figure 1).

Density of CB+/NR2A+ Neurons

The ANOVA performed on layer 2 revealed that the effect of diagnosis was highly significant [F(2,57) = 4.33; p = .018; Figure 2]. Post hoc unpaired t tests further revealed that the density of these neurons was significantly increased in schizophrenia by 41% and 44%, respectively, compared with the normal control (p = .012) and bipolar disorder

Discussion

We have previously found that, in layer 2 of the ACCx, the expression of NR2A mRNA in approximately 53% and 35% of the GABA cells in schizophrenia and bipolar disorder, respectively, seems to be decreased to a level that is no longer experimentally detectable (9). Because CB-containing GABA cells tend to be more heavily localized to layer 2 of the cortex (16, 19, 20), we hypothesized that they would be among the GABA neurons that are affected. Our data suggest that, contrary to this hypothesis,

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Our results implicate cells that typically express GluN2A-rich NMDARs as particularly affected in schizophrenia. Previous studies show less GluN2A-containing inhibitory neurons in the cortex in schizophrenia (Woo et al., 2004; Kinney et al., 2006; Woo et al., 2008b; Bitanihirwe et al., 2009; Alherz et al., 2017). Indeed, our cohort also exhibits reductions in the gene expression of many inhibitory neuron transcripts in DLPFC in schizophrenia (Fung et al., 2010).
Lower N-methyl-d-aspartate receptor (NMDAR) GluN1 subunit levels and heightened neuroinflammation are found in the cortex in schizophrenia. Since neuroinflammation can lead to changes in NMDAR function, it is possible that these observations are linked in schizophrenia. We aimed to extend our previous studies by measuring molecular indices of NMDARs that define key functional properties of this receptor — particularly the ratio of GluN2A and GluN2B subunits — in dorsolateral prefrontal cortex (DLPFC) from schizophrenia and control cases (37/37). We sought to test whether changes in these measures are specific to the subset of schizophrenia cases with high levels of inflammation-related mRNAs, defined as a high inflammatory subgroup. Quantitative autoradiography was used to detect ‘functional’ NMDARs ([³H]MK-801), GluN1-coupled-GluN2A subunits ([³H]CGP-39653), and GluN1-coupled-GluN2B subunits ([³H]Ifenprodil). Quantitative RT-PCR was used to measure NMDAR subunit transcripts (GRIN1, GRIN2A and GRIN2B). The ratios of GluN2A:GluN2B binding and GRIN2A:GRIN2B mRNAs were calculated as an index of putative NMDAR composition. We found: 1) GluN2A binding, and 2) the ratios of GluN2A:GluN2B binding and GRIN2A:GRIN2B mRNAs were lower in schizophrenia cases versus controls (p < 0.05), and 3) lower GluN2A:GluN2B binding and GRIN2A:GRIN2B mRNA ratios were exaggerated in the high inflammation/schizophrenia subgroup compared to the low inflammation/control subgroup (p < 0.05). No other NMDAR-related indices were significantly changed in the high inflammation/schizophrenia subgroup. This suggests that neuroinflammation may alter NMDAR stoichiometry rather than targeting total NMDAR levels overall, and future studies could aim to determine if anti-inflammatory treatment can alleviate this aspect of NMDAR-related pathology.
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Reported alteration in NMDAR function has supported the proposition of hypo-glutamatergic and hyper-glutamatergic states [7,8]. As NMDAR are thought to be important mediators of neural plasticity due to their time-dependent characteristics and their regulation of postsynaptic calcium (Ca2+) concentration, both hyper- and hypo-glutamatergic states have been associated with potential impairments of long-term potentiation (LTP) and long-term depression (LTD) respectively [9–11]. Furthermore, post-mortem studies of brain tissue of schizophrenia patients have consistently demonstrated reductions in glutamic acid decarboxylase (GAD67) and GABAergic interneuron density, suggesting additional impairments in inhibitory regulation [12].
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Increasing evidence suggests that the postsynaptic density (PSD) is a subcellular microdomain in which multiple etiologic factors may converge and impact common final pathways for the pathophysiology of schizophrenia. Housed in dendritic spines, the PSD is the hub of postsynaptic neuronal signaling and consists of macromolecular complexes, in which glutamatergic and other signaling pathways implicated for schizophrenia reside and interact. Genetics studies have shown that common and rare genetic variants are highly enriched among the pathways concentrated in the PSD. Postmortem studies have reported alterations in the expression of various proteins as well as signaling activity in glutamatergic pathways. Recent studies support the notion that multiple molecular abnormalities may converge in macromolecular complexes in the PSD precipitating alterations in N-methyl-d-aspartate receptor signaling. Thus, the proteome of the PSD and protein interactions therein will be an important avenue to investigate mechanisms underlying convergence of multifactorial and heterogeneous etiologies in schizophrenia.
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N-methyl-D-aspartate receptor (NMDAR) hypofunction in parvalbumin-expressing (PV+) inhibitory neurons (INs) may contribute to symptoms in patients with schizophrenia (SZ). This hypothesis was inspired by studies in humans involving NMDAR antagonists that trigger SZ symptoms. Animal models of SZ using neuropharmacology and genetic knockouts have successfully replicated some of the key observations in human subjects involving alteration of gamma band oscillations (GBO) observed in electroencephalography and magnetoencephalography signals. However, it remains to be seen if NMDAR hypofunction in PV+ neurons is fundamental to the phenotype observed in these models. In this review, we discuss some of the key computational models of GBO and their predictions in the context of NMDAR hypofunction in INs. While PV+ INs have been the main focus of SZ studies in animal models, we also discuss the implications of NMDAR hypofunction in other types of INs using computational models for GBO modulation in the visual cortex.
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While the studies reviewed using total homogenate or film-based in situ hybridization techniques suggest that the NR2A subunit of the NMDA receptor is not greatly altered in people with schizophrenia, two studies of NR2A subunit mRNA in interneurons suggest otherwise. Using double-label in situ hybridization to define interneuron populations, Woo et al. (2004); Woo, Shrestha, Lamb, Minns, and Benes (2008) found a decreased density of NR2A mRNA expressing GAD67 positive interneurons, but an increased density in the subset of NR2A and calbindin mRNA-containing neurons in layer II of anterior cingulate cortex. This is noteworthy, as the gene for NR2A has been implicated in the etiology of schizophrenia in large genetics studies (Fromer et al., 2014; Ripke et al., 2014).
Evidence suggests that anomalous mismatch negativity (MMN) in schizophrenia is related to glutamatergic abnormalities, possibly involving N-methyl-d-aspartate (NMDA) receptors. Decreased cortical expressions of NMDA receptor subunits have been observed in schizophrenia, though not consistently. To aid with integration and interpretation of previous work, we performed a meta-analysis of effect sizes of mRNA or protein levels of the obligatory NR1 subunit in prefrontal cortex from people with schizophrenia. In schizophrenia compared to unaffected controls the pooled effect size was −0.64 (95% confidence interval: −1.08 to −0.20) for NR1 mRNA reduction and −0.44 (95% confidence interval: −0.80 to −0.07) for NR1 protein reduction. These results represent the first step to a deeper understanding of the region-specific, cell-specific, and stage-specific NMDA receptor hypofunction in schizophrenia, which could be linked to mismatch negativity deficits via transgenic and pharmacological animal models.

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Archival ReportN-Methyl-D-Aspartate Receptor and Calbindin-Containing Neurons in the Anterior Cingulate Cortex in Schizophrenia and Bipolar Disorder

Background

Methods

Results

Conclusions

Section snippets

Subjects

Statistical Analyses

Density of All CB+ Neurons

Density of CB+/NR2A+ Neurons

Discussion

Schizophrenia genes, gene expression, and neuropathology: On the matter of their convergence

Mol Psychiatry

The emerging role of glutamate in the pathophysiology and treatment of schizophrenia

Am J Psychiatry

The glutamatergic dysfunction hypothesis for schizophrenia

Harv Rev Psychiatry

Converging evidence of NMDA receptor hypofunction in the pathophysiology of schizophrenia

Ann N Y Acad Sci

Molecular abnormalities of the glutamate synapse in the thalamus in schizophrenia

Ann N Y Acad Sci

Glutamate as a therapeutic target in psychiatric disorders

Mol Psychiatry

Glutamate receptor dysfunction and schizophrenia

Arch Gen Psychiatry

NMDA receptor hypofunction model of schizophrenia

J Psychiatr Res

Density of glutamic acid decarboxylase 67 messenger RNA-containing neurons that express the N-methyl-D-aspartate receptor subunit NR2A in the anterior cingulate cortex in schizophrenia and bipolar disorder

Arch Gen Psychiatry

Organizing principles for a diversity of GABAergic interneurons and synapses in the neocortex

Science

Division of labor among distinct subtypes of inhibitory neurons in a cortical microcircuit of working memory

Proc Natl Acad Sci U S A

Interneurons of the neocortical inhibitory system

Nat Rev Neurosci

Molecular and physiological diversity of cortical nonpyramidal cells

J Neurosci

Neocortical neuronal diversity: Chemical heterogeneity revealed by colocalization studies of classic neurotransmitters, neuropeptides, calcium-binding proteins, and cell surface molecules

Cereb Cortex

GABAergic neuronal subtypes in the human frontal cortex—development and deficits in schizophrenia

J Chem Neuroanat

Types of neurons, synaptic connections and chemical characteristics of cells immunoreactive for calbindin-D28K, parvalbumin and calretinin in the neocortex

J Chem Neuroanat

Local circuit neurons immunoreactive for calretinin, calbindin D-28k or parvalbumin in monkey prefrontal cortex: Distribution and morphology

J Comp Neurol

GABAergic cell subtypes and their synaptic connections in rat frontal cortex

Cereb Cortex

The density and spatial distribution of GABAergic neurons, labelled using calcium binding proteins, in the anterior cingulate cortex in major depressive disorder, bipolar disorder, and schizophrenia

Biol Psychiatry

Archival Report
N-Methyl-D-Aspartate Receptor and Calbindin-Containing Neurons in the Anterior Cingulate Cortex in Schizophrenia and Bipolar Disorder