Pathological Basis for Deficient Excitatory Drive to Cortical Parvalbumin Interneurons in Schizophrenia.
ABSTRACT: Deficient excitatory drive to parvalbumin-containing cortical interneurons is proposed as a key neural substrate for altered gamma oscillations and cognitive dysfunction in schizophrenia. However, a pathological entity producing such a deficit has not been identified. The authors tested the hypothesis that cortical parvalbumin interneurons receive fewer excitatory synaptic inputs in individuals with schizophrenia.Fluorescent immunohistochemistry, confocal microscopy, and post-image processing techniques were used to quantify the number of putative excitatory synapses (i.e., the overlap of vesicular glutamate transporter 1-positive [VGlut1+] puncta and postsynaptic density protein 95-positive [PSD95+] puncta) per surface area of parvalbumin-positive (PV+) or calretinin-positive (CR+) neurons in the dorsolateral prefrontal cortex from schizophrenia subjects and matched unaffected comparison subjects.Mean density of VGlut1+/PSD95+ puncta on PV+ neurons was 18% lower in schizophrenia, a significant difference. This deficit was not influenced by methodological confounds or schizophrenia-associated comorbid factors, not present in monkeys chronically exposed to antipsychotic medications, and not present in CR+ neurons. Mean density of VGlut1+/PSD95+ puncta on PV+ neurons predicted the activity-dependent expression levels of parvalbumin and glutamic acid decarboxylase 67 (GAD67) in schizophrenia subjects but not comparison subjects.To the authors' knowledge, this is the first demonstration that excitatory synapse density is lower selectively on parvalbumin interneurons in schizophrenia and predicts the activity-dependent down-regulation of parvalbumin and GAD67. Because the activity of parvalbumin interneurons is required for generation of cortical gamma oscillations and working memory function, these findings reveal a novel pathological substrate for cortical dysfunction and cognitive deficits in schizophrenia.
Project description:Markers of GABA neurotransmission are altered in multiple regions of the neocortex in individuals with schizophrenia. Lower levels of glutamic acid decarboxylase 67 (GAD67) mRNA and protein, which is responsible for most cortical GABA synthesis, are accompanied by lower levels of GABA membrane transporter 1 (GAT1) mRNA. These alterations are thought to be most prominent in the parvalbumin (PV)-containing subclass of interneurons, which also contain lower levels of PV mRNA. Since GAT1 and PV each reduce the availability of GABA at postsynaptic receptors, lower levels of GAT1 and PV mRNAs have been hypothesized to represent compensatory responses to an upstream reduction in cortical GABA synthesis in schizophrenia. However, such cause-and-effect hypotheses cannot be directly tested in a human illness. Consequently, we used two mouse models with reduced GAD67 expression specifically in PV neurons (PV(GAD67+/-)) or in all interneurons (GABA(GAD67+/-)) and quantified GAD67, GAT1 and PV mRNA levels using methods identical to those employed in studies of schizophrenia. Cortical levels of PV or GAT1 mRNAs were not altered in PV(GAD67+/-) mice during postnatal development or in adulthood. Furthermore, cellular analyses confirmed the predicted reduction in GAD67 mRNA, but failed to show a deficit in PV mRNA in these animals. Levels of PV and GAT1 mRNAs were also unaltered in GABA(GAD67+/-) mice. Thus, mouse lines with cortical reductions in GAD67 mRNA that match or exceed those present in schizophrenia, and that differ in the developmental timing and cell type-specificity of the GAD67 deficit, failed to provide proof-of-concept evidence that lower PV and GAT1 expression in schizophrenia are a consequence of lower GAD67 expression. Together, these findings suggest that the correlated decrements in cortical GAD67, PV and GAT1 mRNAs in schizophrenia may be a common consequence of some other upstream factor.
Project description:Cognitive processing is highly dependent on the functional integrity of gamma-amino-butyric acid (GABA) interneurons in the brain. These cells regulate excitability and synaptic plasticity of principal neurons balancing the excitatory/inhibitory tone of cortical networks. Reduced function of parvalbumin (PV) interneurons and disruption of GABAergic synapses in the cortical circuitry result in desynchronized network activity associated with cognitive impairment across many psychiatric disorders, including schizophrenia. However, the mechanisms underlying these complex phenotypes are still poorly understood. Here we show that in animal models, genetic deletion of fibroblast growth factor 14 (Fgf14), a regulator of neuronal excitability and synaptic transmission, leads to loss of PV interneurons in the CA1 hippocampal region, a critical area for cognitive function. Strikingly, this cellular phenotype associates with decreased expression of glutamic acid decarboxylase 67 (GAD67) and vesicular GABA transporter (VGAT) and also coincides with disrupted CA1 inhibitory circuitry, reduced in vivo gamma frequency oscillations and impaired working memory. Bioinformatics analysis of schizophrenia transcriptomics revealed functional co-clustering of FGF14 and genes enriched within the GABAergic pathway along with correlatively decreased expression of FGF14, PVALB, GAD67 and VGAT in the disease context. These results indicate that Fgf14(-/-) mice recapitulate salient molecular, cellular, functional and behavioral features associated with human cognitive impairment, and FGF14 loss of function might be associated with the biology of complex brain disorders such as schizophrenia.
Project description:Human postmortem studies suggest a major role for abnormalities in GABAergic interneurons in the prefrontal cortex in schizophrenia. Cortical interneurons differentiated from induced pluripotent stem cells (iPSCs) of schizophrenia subjects showed significantly lower levels of glutamate decarboxylase 67 (GAD67), replicating findings from multiple postmortem studies, as well as reduced levels of synaptic proteins gehpyrin and NLGN2. Co-cultures of the interneurons with excitatory cortical pyramidal neurons from schizophrenia iPSCs showed reduced synaptic puncta density and lower action potential frequency. NLGN2 overexpression in schizophrenia neurons rescued synaptic puncta deficits while NLGN2 knockdown in healthy neurons resulted in reduced synaptic puncta density. Schizophrenia interneurons also had significantly smaller nuclear area, suggesting an innate oxidative stressed state. The antioxidant N-acetylcysteine increased the nuclear area in schizophrenia interneurons, increased NLGN2 expression and rescued synaptic deficits. These results implicate specific deficiencies in the synaptic machinery in cortical interneurons as critical regulators of synaptic connections in schizophrenia and point to a nexus between oxidative stress and NLGN2 expression in mediating synaptic deficits in schizophrenia.
Project description:Dysfunction of cortical parvalbumin (PV)-containing GABAergic interneurons has been implicated in cognitive deficits of schizophrenia. In humans microdeletion of the CHRNA7 (?7 nicotinic acetylcholine receptor, nAChR) gene is associated with cortical dysfunction in a broad spectrum of neurodevelopmental and neuropsychiatric disorders including schizophrenia while in mice similar deletion causes analogous abnormalities including impaired attention, working-memory and learning. However, the pathophysiological roles of ?7 nAChRs in cortical PV GABAergic development remain largely uncharacterized. In both in vivo and in vitro models, we identify here that deletion of the ?7 nAChR gene in mice impairs cortical PV GABAergic development and recapitulates many of the characteristic neurochemical deficits in PV-positive GABAergic interneurons found in schizophrenia. ?7 nAChR null mice had decreased cortical levels of GABAergic markers including PV, glutamic acid decarboxylase 65/67 (GAD65/67) and the ?1 subunit of GABAA receptors, particularly reductions of PV and GAD67 levels in cortical PV-positive interneurons during late postnatal life and adulthood. Cortical GABAergic synaptic deficits were identified in the prefrontal cortex of ?7 nAChR null mice and ?7 nAChR null cortical cultures. Similar disruptions in development of PV-positive GABAergic interneurons and perisomatic synapses were found in cortical cultures lacking ?7 nAChRs. Moreover, NMDA receptor expression was reduced in GABAergic interneurons, implicating NMDA receptor hypofunction in GABAergic deficits in ?7 nAChR null mice. Our findings thus demonstrate impaired cortical PV GABAergic development and multiple characteristic neurochemical deficits reminiscent of schizophrenia in cortical PV-positive interneurons in ?7 nAChR gene deletion models. This implicates crucial roles of ?7 nAChRs in cortical PV GABAergic development and dysfunction in schizophrenia and other neuropsychiatric disorders.
Project description:We have previously reported that the expression of the messenger ribonucleic acid (mRNA) for the NR2A subunit of the N-methyl-D-aspartate (NMDA) class of glutamate receptor was decreased in a subset of inhibitory interneurons in the cerebral cortex in schizophrenia. In this study, we sought to determine whether a deficit in the expression of NR2A mRNA was present in the subset of interneurons that contain the calcium buffer parvalbumin (PV) and whether this deficit was associated with a reduction in glutamatergic inputs in the prefrontal cortex (PFC) in schizophrenia.We examined the expression of NR2A mRNA, labeled with a 35S-tagged riboprobe, in neurons that expressed PV mRNA, visualized with a digoxigenin-labeled riboprobe via an immunoperoxidase reaction, in twenty schizophrenia and twenty matched normal control subjects. We also immunohistochemically labeled the glutamatergic axon terminals with an antibody against vGluT1.The density of the PV neurons that expressed NR2A mRNA was significantly decreased by 48-50% in layers 3 and 4 in the subjects with schizophrenia, but the cellular expression of NR2A mRNA in the PV neurons that exhibited a detectable level of this transcript was unchanged. In addition, the density of vGluT1-immunoreactive boutons was significantly decreased by 79% in layer 3, but was unchanged in layer 5 of the PFC in schizophrenia.These findings suggest that glutamatergic neurotransmission via NR2A-containing NMDA receptors on PV neurons in the PFC may be deficient in schizophrenia. This may disinhibit the postsynaptic excitatory circuits, contributing to neuronal injury, aberrant information flow and PFC functional deficits in schizophrenia.
Project description:Decreased expression of dopamine D2 receptors (D2R), dysfunction of inhibitory neurotransmission and impairments in the structure and connectivity of neurons in the medial prefrontal cortex (mPFC) are involved in the pathogenesis of schizophrenia and major depression, but the relationship between these changes remains unclear. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM), a plasticity-related molecule, may serve as a link. This molecule is expressed in cortical interneurons and dopamine, via D2R, modulates its expression in parallel to that of proteins related to synapses and inhibitory neurotransmission, suggesting that D2R-targeted antipsychotics/antidepressants may act by affecting the plasticity of mPFC inhibitory circuits. To understand the role of PSA-NCAM in this plasticity, rats were chronically treated with a D2R agonist (PPHT) after cortical PSA depletion. PPHT-induced increases in GAD67 and synaptophysin (SYN) neuropil expression were blocked when PSA was previously removed, indicating a role for PSA-NCAM in this plasticity. The number of PSA-NCAM expressing interneuron somata also increased after PPHT treatment, but the percentages of these cells belonging to different interneuronal subpopulations did not change. Cortical pyramidal neurons did not express PSA-NCAM, but puncta co-expressing this molecule and parvalbumin could be found surrounding their somata. PPHT treatment increased the number of PSA-NCAM and parvalbumin expressing perisomatic puncta, but decreased the percentage of parvalbumin puncta that co-expressed SYN. PSA depletion did not block these effects on the perisomatic region, but increased further the number of parvalbumin expressing puncta and increased the percentage of puncta co-expressing SYN and parvalbumin, suggesting that the polysialylation of NCAM may regulate perisomatic inhibition of mPFC principal neurons. Summarizing, the present results indicate that dopamine acting on D2R influences structural plasticity of mPFC interneurons and point to PSA-NCAM as a key player in this remodeling.
Project description:Decreased expression of the GABA synthetic enzyme glutamate decarboxylase 67 (GAD67) in a subset of GABAergic neurons, including parvalbumin (PV)-expressing neurons, has been observed in postmortem brain studies of schizophrenics and in animal models of schizophrenia. However, it is unclear whether and how the perturbations of GAD67-mediated GABA synthesis and signaling contribute to the pathogenesis of schizophrenia. To address this issue, we generated the mice lacking GAD67 primarily in PV neurons and characterized them with focus on schizophrenia-related parameters. We found that heterozygous mutant mice exhibited schizophrenia-related behavioral abnormalities such as deficits in prepulse inhibition, MK-801 sensitivity, and social memory. Furthermore, we observed reduced inhibitory synaptic transmission, altered properties of NMDA receptor-mediated synaptic responses in pyramidal neurons, and increased spine density in hippocampal CA1 apical dendrites, suggesting a possible link between GAD67 deficiency and disturbed glutamatergic excitatory synaptic functions in schizophrenia. Thus, our results indicate that the mice heterozygous for GAD67 deficiency primarily in PV neurons share several neurochemical and behavioral abnormalities with schizophrenia, offering a novel tool for addressing the underlying pathophysiology of schizophrenia.
Project description:Disrupted-in-Schizophrenia-1 (DISC1) gene has been linked to schizophrenia and related major mental illness. Mouse Disc1 has been implicated in brain development, mainly in the proliferation, differentiation, lamination, neurite outgrowth and synapse formation and maintenance of cortical excitatory neurons. Here, the effects of two loss-of-function point mutations in the mouse Disc1 sequence (Q31L and L100P) on cortical inhibitory interneurons were investigated. None of the mutations affected the overall number of interneurons. However, the 100P, but not the 31L, mutation resulted in a significant decrease in the numbers of interneurons expressing parvalbumin mRNA and protein across the sensory cortex. To investigate role of Disc1 in regulation of parvalbumin expression, mouse wild-type Disc-1 or the 100P mutant form were electroporated in utero into cortical excitatory neurons. Overexpression of wild-type Disc1 in these cells caused increased densities of parvalbumin-expressing interneurons in the electroporated area and in areas connected with it, whereas expression of Disc1-100P did not. We conclude that the 100P mutation prevents expression of parvalbumin by a normally sized cohort of interneurons and that altering Disc1 function in cortical excitatory neurons indirectly affects parvalbumin expression by cortical interneurons, perhaps as a result of altered functional input from the excitatory neurons.
Project description:Exposure to maternal stress (MS) and mutations in GAD1, which encodes the ?-aminobutyric acid (GABA) synthesizing enzyme glutamate decarboxylase (GAD) 67, are both risk factors for psychiatric disorders. However, the relationship between these risk factors remains unclear. Interestingly, the critical period of MS for psychiatric disorders in offspring corresponds to the period of GABAergic neuron neurogenesis and migration in the fetal brain, that is, in the late stage of gestation. Indeed, decrement of parvalbumin (PV)-positive GABAergic interneurons in the medial prefrontal cortex (mPFC) and hippocampus (HIP) has often been observed in schizophrenia patients. In the present study, we used GAD67-green fluorescent protein (GFP) knock-in mice (that is, mice in which the Gad1 gene is heterozygously deleted; GAD67(+/GFP)) that underwent prenatal stress from embryonic day 15.0 to 17.5 and monitored PV-positive GABAergic neurons to address the interaction between Gad1 disruption and stress. Administration of 5-bromo-2-deoxyuridine revealed that neurogenesis of GFP-positive GABAergic neurons, but not cortical plate cells, was significantly diminished in fetal brains during MS. Differential expression of glucocorticoid receptors by different progenitor cell types may underlie this differential outcome. Postnatally, the density of PV-positive, but not PV-negative, GABAergic neurons was significantly decreased in the mPFC, HIP and somatosensory cortex but not in the motor cortex of GAD67(+/GFP) mice. By contrast, these findings were not observed in wild-type (GAD67(+/+)) offspring. These results suggest that prenatal stress, in addition to heterozygous deletion of Gad1, could specifically disturb the proliferation of neurons destined to be PV-positive GABAergic interneurons.
Project description:Subclasses of ?-aminobutyric acid (GABA) interneurons differentially influence cortical network activity. The contribution of differences in GABA synthesis and reuptake in axon boutons to cell type-specific functions is unknown. GABA is synthesized within boutons by glutamic acid decarboxylase 65 (GAD65) and GAD67, while GAT1 is responsible for GABA reuptake. Using an imaging methodology capable of determining the colocalization frequency of different immunocytochemical labels in the same bouton and the quantification of the fluorescence intensity of each label in these same structures, we assessed the bouton levels of GAD65, GAD67, and GAT1 in parvalbumin-expressing chandelier (PV(ch)) and basket (PV(b)) neurons and cannabinoid 1 receptor-expressing basket (CB1r(b)) neurons in the monkey prefrontal cortex. We show that PV(ch) boutons almost exclusively contained GAD67, relative to GAD65, whereas CB1r(b) boutons contained mostly GAD65. In contrast, both GAD65 and GAD67 were easily detected in PV(b) boutons. Furthermore, in comparison with PV(ch) boutons, CB1r(b) boutons expressed low to undetectable levels of GAT1. Our findings provide a new basis for the distinctive functional roles of these perisomatic-innervating interneurons in cortical circuits. In addition, they strongly suggest that altered levels of GAD67 or GAD65, as seen in some psychiatric diseases, would have cell type-specific consequences on the modulation of GABA neurotransmission.