Restoration of Sp4 in Forebrain GABAergic Neurons Rescues Hypersensitivity to Ketamine in Sp4 Hypomorphic Mice.

BackgroundKetamine produces schizophrenia-like behavioral phenotypes in healthy people. Prolonged ketamine effects and exacerbation of symptoms after the administration of ketamine have been observed in patients with schizophrenia. More recently, ketamine has been used as a potent antidepressant to treat patients with major depression. The genes and neurons that regulate behavioral responses to ketamine, however, remain poorly understood. Sp4 is a transcription factor for which gene expression is restricted to neuronal cells in the brain. Our previous studies demonstrated that Sp4 hypomorphic mice display several behavioral phenotypes relevant to psychiatric disorders, consistent with human SP4 gene associations with schizophrenia, bipolar disorder, and major depression. Among those behavioral phenotypes, hypersensitivity to ketamine-induced hyperlocomotion has been observed in Sp4 hypomorphic mice.MethodsIn the present study, we used the Cre-LoxP system to restore Sp4 gene expression, specifically in either forebrain excitatory or GABAergic inhibitory neurons in Sp4 hypomorphic mice. Mouse behavioral phenotypes related to psychiatric disorders were examined in these distinct rescue mice.ResultsRestoration of Sp4 in forebrain excitatory neurons did not rescue deficient sensorimotor gating nor ketamine-induced hyperlocomotion. Restoration of Sp4 in forebrain GABAergic neurons, however, rescued ketamine-induced hyperlocomotion, but did not rescue deficient sensorimotor gating.ConclusionsOur studies suggest that the Sp4 gene in forebrain GABAergic neurons regulates ketamine-induced hyperlocomotion

System-Wide Immunohistochemical Analysis of Protein Co-Localization

Background: The analysis of co-localized protein expression in a tissue section is often conducted with immunofluorescence histochemical staining which is typically visualized in localized regions. On the other hand, chromogenic immunohistochemical staining, in general, is not suitable for the detection of protein co-localization. Here, we developed a new protocol, based on chromogenic immunohistochemical stain, for system-wide detection of protein co-localization and differential expression. Methodology/Principal Findings: In combination with a removable chromogenic stain, an efficient antibody stripping method was developed to enable sequential immunostaining with different primary antibodies regardless of antibody’s host species. Sections were scanned after each staining, and the images were superimposed together for the detection of protein co-localization and differential expression. As a proof of principle, differential expression and co-localization of glutamic acid decarboxylase67 (GAD67) and parvalbumin proteins was examined in mouse cortex. Conclusions/Significance: All parvalbumin-containing neurons express GAD67 protein, and GAD67-positive neurons that do not express parvalbumin were readily visualized from thousands of other neurons across mouse cortex. The method provided a global view of protein co-localization as well as differential expression across an entire tissue section. Repeate

A novel animal model for neuroinflammation and white matter degeneration

Small interference RNA has been widely used to suppress gene expression. Three different short hairpin RNAs (shRNAs) against dopamine D1 receptor (Drd1), driven by mouse U6 promoter in self-complementary AAV8 vector (scAAV8), were used to silence mouse striatal Drd1 expression. Transduction of mouse striatum with all three scAAV8-D1shRNA viruses, but not the control scAAV8 virus, causes extensive neuroinflammation, demyelination, and axon degeneration. RNA interference is known to be coupled to the innate immune system as a host cell defense against virus infection. Activation of the innate immune system may play a causal role in the development of neuroinflammation and white matter degeneration, providing a novel animal model for multiple sclerosis (MS) and other neuroinflammatory diseases

Over-expression of XIST, the Master Gene for X Chromosome Inactivation, in Females With Major Affective Disorders

Background: Psychiatric disorders are common mental disorders without a pathological biomarker. Classic genetic studies found that an extra X chromosome frequently causes psychiatric symptoms in patients with either Klinefelter syndrome (XXY) or Triple X syndrome (XXX). Over-dosage of some X-linked escapee genes was suggested to cause psychiatric disorders. However, relevance of these rare genetic diseases to the pathogenesis of psychiatric disorders in the general population of psychiatric patients is unknown. Methods: XIST and several X-linked genes were studied in 36 lymphoblastoid cell lines from healthy females and 60 lymphoblastoid cell lines from female patients with either bipolar disorder or recurrent major depression. XIST and KDM5C expression was also quantified in 48 RNA samples from postmortem human brains of healthy female controls and female psychiatric patients. Findings: We found that the XIST gene, a master in control of X chromosome inactivation (XCI), is significantly over-expressed (p = 1 × 10−7, corrected after multiple comparisons) in the lymphoblastoid cells of female patients with either bipolar disorder or major depression. The X-linked escapee gene KDM5C also displays significant up-regulation (p = 5.3 × 10−7, corrected after multiple comparisons) in the patients' cells. Expression of XIST and KDM5C is highly correlated (Pearson's coefficient, r = 0.78, p = 1.3 × 10−13). Studies on human postmortem brains supported over-expression of the XIST gene in female psychiatric patients. Interpretations: We propose that over-expression of XIST may cause or result from subtle alteration of XCI, which up-regulates the expression of some X-linked escapee genes including KDM5C. Over-expression of X-linked genes could be a common mechanism for the development of psychiatric disorders between patients with those rare genetic diseases and the general population of female psychiatric patients with XIST over-expression. Our studies suggest that XIST and KDM5C expression could be used as a biological marker for diagnosis of psychiatric disorders in a significantly large subset of female patients. Research in context: Due to lack of biological markers, diagnosis and treatment of psychiatric disorders are subjective. There is utmost urgency to identify biomarkers for clinics, research, and drug development. We found that XIST and KDM5C gene expression may be used as a biological marker for diagnosis of major affective disorders in a significantly large subset of female patients from the general population. Our studies show that over-expression of XIST and some X-linked escapee genes may be a common mechanism for development of psychiatric disorders between the patients with rare genetic diseases (XXY or XXX) and the general population of female psychiatric patients

Chromogenic immunohistochemical analysis of GAD67 and parvalbumin in mouse cortex.

<p>Mouse brain paraffin section was first used for anti-GAD67 immunostaining. After the stained slide was scanned with Aperio ScanScope, the anti-GAD67 primary antibody and red stain color were completely stripped. The same section was re-used for anti-parvalbumin immunostaining.</p

Multiple rounds of sequential immunohistochemical analysis.

<p>Mouse hippocampus and cortex were immunostained with 5 different antibodies: (<b>a</b>) anti-GAD67, high expression of GAD67 in hippocampal mossy fiber besides in cortex, (<b>b</b>) anti-parvalbumin, no detectable parvalbumin in hippocampal mossy fiber, but abundant in cortex, (<b>c</b>) anti-calretinin, sporadic calretinin positive cells in cortex with abundant calretinin expression in the inner molecular layer of dentate gyrus, (<b>d</b>) anti-Iba-1, numerous microglial cells and their processes across the section, (<b>e</b>) anti-MBP, abundant myelin in <i>corpus callosum</i> and some myelinated neurons in hippocampus and cortex. Scale bar: 500 µm.</p

Co-localization between GAD67, parvalbumin, and calretinin.

<p>Top panel: the images of GAD67, parvalbumin, and calretinin were pseudocolored into green, red, and purple respectively. Bottom panel: the three images were superimposed together. Arrow 1: calretinin positive neuron. Arrow 2: GAD67 positive neuron. Arrow 3: neuron expressing both GAD67 and parvalbumin. Arrow 4: neuron expressing both GAD67 and calretinin. Scale bar: 500 µm.</p

Parvalbumin and GAD67 co-localization.

<p>(<b>a</b>) The color of the anti-parvalbumin image was inverted and further superimposed onto the anti-GAD67 image (bottom). Parvalbumin expression only cells would appear as bright spots. However, there were no parvalbumin-positive cells that did not express GAD67. Scale bar: 500 µm. Deep layers (A) and the middle layers (B) were visualized at a higher magnification (<b>b</b>). GAD67 expression only neurons became darker spots in deep layers (A) of frontal cortex (black arrow heads). Co-localization between GAD67 and parvalbumin in the middle layers (B) of motor cortex (red arrow heads). Scale bar: 100 µm.</p