Elsevier

Brain Research

Volume 1392, 25 May 2011, Pages 47-53
Brain Research

Research Report
Impairment of the tyrosine hydroxylase neuronal network in the orbitofrontal cortex of a genetically modified mouse model of schizophrenia

https://doi.org/10.1016/j.brainres.2011.03.058Get rights and content

Abstract

Important genes have been identified that are associated with susceptibility to schizophrenia. DISC1 is one of these candidate genes. The protein 14-3-3 epsilon is a DISC1-interacting molecule and is associated with axon elongation. The genetically modified 14-3-3 epsilon heterozygous knockout mice are considered to be an animal model of schizophrenia because they present endophenotypes of schizophrenia including working memory impairment. This study investigated the immunohistochemical expression of tyrosine hydroxylase (TH) to reveal the alterations in the functional structure of the axon elongation caused by the deficit of 14-3-3 epsilon. The study focused on the orbitofrontal cortex in the prefrontal cortex which is a region of interest in schizophrenia research. The investigation used eight 15-week-old knockout mice and six age-matched wild-type mice. The TH immunopositive fibers were linear and dense in the wild-type mice. These fibers were serpentine, thin and short in the knockout mice. Although it appeared that dendritic spine-like immunopositive varices were strung tightly in the fibers of wild-type mice, these were few and sparse in those of the of the knockout mice. Quantitative analysis showed a significant decrease in the total extent of the TH-immunopositive fibers in the orbital cortex of the knockout mouse. There is thought to be a dysfunction of a neurotransmitter such as dopamine and noradrenalin in the prefrontal cortex of these knockout mice.

Research highlights

► Impairment of the orbitofrontal cortex of a 14-3-3 epsilon heterozygous knockout mouse. ► Immunohistochemical approach to reveal the alterations in the functional structure. ► A significant decrease of tyrosine hydroxylase immunopositive fibers in this area. ► Few and sparse dendritic spine-like varices of tyrosine hydroxylase immunopositive fibers.

Introduction

Schizophrenia is a common neuropsychiatric disorder that occurs in approximately 1% of the general population. Although the etiology of schizophrenia remains unclear, it is widely thought to be a neurodevelopment disorder. Many important findings have been obtained by studies of neurochemistry, neurophysiology and neuroimaging. However, alterations in the schizophrenia brain and the etiology of schizophrenia brain and especially the morphology of neuronal fibers must be conducted by direct observation of the brain tissue itself by microscopy.

Recently, molecular biological investigations have identified several putative schizophrenic candidate genes, and most of these genes are associated with the formation of neuronal networks, expanding neuronal fibers, migration of neurons, etc. (Harrison, 2007, Iritani, 2007). One of the major candidate genes is disrupted-in-schizophrenia 1 (DISC1). This is a promising candidate susceptibility gene for schizophrenia, which was first described as a strong candidate gene in a large Scottish family in which a balanced chromosomal translocation segregates with schizophrenia and other psychiatric disorders (Blackwood et al., 2001, Brandon et al., 2009, Chubb et al., 2008). DISC1 is also involved in neurodevelopment, including axonal elongation. In this process, DISC1 interacts with a complex formed by NudE-like (NUDEL), lissencephaly-1 (LIS1) and 14-3-3 epsilon (Taya et al., 2007).

The 14-3-3 epsilon protein is a member of the 14-3-3 family and is also one of the DISC1-interacting molecules. The 14-3-3 proteins are ubiquitous proteins that are highly conserved from bacteria to humans and plants and have several molecular and cellular functions such as signal transduction, cell cycle regulation, apoptosis and stress responses (Fu et al., 2000, Muslin and Xing, 2000, Tzivion and Avruch, 2002). There are seven distinct mammalian isoforms of 14-3-3 proteins, and some of these were previously reported to have a genetic association with schizophrenia (Toyooka et al., 1999, Wong et al., 2003, Wong et al., 2005).

YWHAE, a gene encoding 14-3-3 epsilon, was reported to be a susceptibility gene for schizophrenia and genetically modified 14-3-3 epsilon heterozygous knockout (KO) mice present various endophenotypes of schizophrenia including working memory deficits or cognitive decline (Ikeda et al., 2008). Therefore, this 14-3-3 epsilon heterozygous KO mouse is considered to be a novel animal model for schizophrenia.

Tyrosine hydroxylase (TH) is one of catecholaminergic markers (Beeler et al., 2009, Nair-Roberts et al., 2008) and is the enzyme responsible for catalyzing the conversion of the amino acid l-tyrosine to catecholamines such as dopamine and noradrenalin, which are transmitters that are closely associated with the pathophysiology of schizophrenia (Howes and Kapur, 2009, Yamamoto and Hornykiewicz, 2004).

This study investigated the immunohistocheminal expression of TH in the brains of the KO mouse using a neuropathological approach to reveal the alterations in the functional structure of the axon elongation caused by the deficit of 14-3-3 epsilon.

The study focused on the orbitofrontal cortex in the prefrontal cortex which is a region of interest in schizophrenia research, because there are several reports noting a volume reduction in the orbitofrontal cortex of schizophrenia patients (Fornito et al., 2009, Nakamura et al., 2008). The ventral tegmental area (VTA) and locus coeruleus (LC) were also observed because they are the nuclei from which the TH neuronal network originates.

Section snippets

Orbital cortex

The immunopositive fibers were linear, dense, orderly and well developed in the wild-type mice. However, these fibers were serpentine, thin and short in the KO mice. Although it appeared that dendritic spine-like immunopositive varices were strung tightly and thick in the fibers of wild-type mice, these were few and sparse in those of the KO mice. The immunopositive neuronal fibers for the two different primary antibodies showed a similar staining pattern. In addition, TH immunoreactivity was

Discussion

This study investigated the changes of TH expression in the orbitofrontal cortex located in the prefrontal cortex, and the VTA and LC of catecholamine-originating neurons in 14-3-3 epsilon heterozygous KO mice.

Although there was no significant difference in the density and size of the neurons in the VTA and LC between the wild-type and KO mice, there was a markedly significant reduction of TH-immunopositive fibers in the orbitofrontal cortex of these projective terminations in the KO mice in

Limitation

There are three limitations in this study. First, to evaluate the true length of a neuronal fiber, the three-dimensional measurement of the length from the soma to the terminal of a neuron is necessary. However, it is impossible to achieve this measurement by using two-dimensional histological experiments. Therefore, we considered that the neuronal fibers observed in an area of pre-determined thickness represented the density of the neuronal network in the limited area. Second, we observed that

Subjects (animal model)

Eight 15-week-old 14-3-3 epsilon heterozygous KO (+/−) mice and the same number of age-matched 14-3-3 epsilon (+/+) littermate (wild-type) mice were used, and these mice were bred under the same conditions. The background of this animal model has been described in previous studies (Ikeda et al., 2008, Toyo-oka et al., 2003).

Preparation

The animals were placed under deep anesthesia by injection of sodium pentobarbital (40 mg/kg body weight i.p.) and then perfused with a tissue fixative solution (4%

Acknowledgment

This research was supported by Grants-in-Aid for Scientific Research (20591400) from the Japan Society for the Promotion of Science (2008–2010).

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