Materials and methods

Immunohistochemistry on paraffin-embedded sections

Representative sections (6 m, fixed in 10% formal saline and processed to paraffin wax) through the brains of adult mice were rehydrated through graded alcohols to H₂O. Sections were incubated in 1% H₂O₂ for 30 min. Nonspecific binding was blocked by incubation with 5% normal goat serum (NGS) for 1 h, which was replaced with D27 anti-serum and incubated overnight at 4°C in a saturated humidity chamber. Antibody binding was demonstrated using an Optimax™ automated immunostainer with the anti-rabbit ABC Elite™ system, diaminobenzidine as the chromogenic substrate and counterstained in Mayers haematoxylin. Secondary anti-rabbit antibody from the kit was applied at 1 : 200 dilution. Sections were dehydrated, mounted with DPX and examined using light microscopy.

To detect DISC1 immunoreactivity (-ir) a specific rabbit polyclonal anti-DISC1 antibody was used at 1 : 500 dilution. DISC1 antipeptide antibody was raised against residues 734–753 of the mouse sequence and affinity purified by Affiniti Research Products, Exeter, UK. This antibody (D27) has been previously described.¹ Specific immune reactivity of antisera was ascertained using dot blot analysis as described previously.^{1, 27} In order to provide further controls for D27 antibody and to demonstrate its specificity, preabsorbtion with a peptide was performed. D27 antibody was preincubated for 4 h with a 10 excess of immunizing peptide before use.

Immunohistochemistry on frozen brain sections

Mice were terminally anaesthetized with IP pentobarbital and transcardially perfused with saline (100 ml) followed by 4% paraformaldehyde (PFA) (100 ml) in 0.1 M phosphate buffer. Brains were removed, postfixed in 4% PFA overnight at 4°C and then transferred to 30% sucrose for cryoprotection. After refrigeration in sucrose for 48 h, brains were frozen and sectioned at 40 m. Free-floating sections were incubated in 30% H₂O₂ for 10 min to reduce endogenous peroxidase activity and then in 1% bovine serum albumin (BSA), 0.3% Triton-X100 blocking buffer to reduce nonspecific background. Sections were then processed for immunostaining using D27 antibody. Sections were incubated in anti-DISC1 antibody, 1 : 250, overnight at 4°C and then stained for 1 h at room temperature (RT) with cy-3-conjugated secondary antiserum, 1 : 100 (Jackson ImmunoResearch). For double labeling, the following antibodies were used: mouse monoclonal anti-MAP2 antibody, 1 : 500 (Sigma), mouse monoclonal anti-GFAP, 1 : 250 (Chemicon), mouse monoclonal anti-Calbindin, 1 : 500 (Abcam Ltd.), mouse monoclonal anti-GAD65, 1 : 500 (BD Biosciences), mouse monoclonal anti-Glutamate transporter (EAAC1), 1 : 500 (Chemicon), mouse monoclonal anti-Gephrin (MA7a), 1 : 500 (Connex). Antibodies were diluted in blocking buffer and applied for 2 h at RT. FITC-conjugated secondary antibody was used at 1 : 100 for 1 h at RT. After extensive washes in PBS, sections were mounted using Vectashield mounting medium (Vector Labs). Images were collected on Leica confocal microscope using Leica TCS NT 'Image' program version 1.6.587

Tissue preparation and western blotting

Cortex from adult mouse brains or whole brain from embryos were used for the developmental profile of DISC1 protein by Western blot analyses. Cortex from three adult mice of the same age were pooled together to make a single sample at certain time point. Alternatively, brains from embryos at the same gestation period obtained from two to three pregnant female mice (approximately 8–10 embryos per mother) were pooled together to represent one particular developmental time point. Tissues were homogenized in sucrose homogenizing buffer (20 mM HEPES, 0.32 M sucrose, 2 l/ml protease inhibitor cocktail, Sigma). Owing to embryo size at E10.5, whole embryos were processed. Lysates were centrifuged at 6000 rpm for 10 min. Lysate protein concentration was quantitated by BCA analysis (Pierce). Samples were then separated by SDS-PAGE, transferred to nitrocellulose membrane (Invitrogen) and blocked in Odyssey blocking buffer (Li-COR) overnight at 4°C. The blocked membrane was incubated with primary antibody (D27 or -tubulin, 1 : 4000, Sigma) for 1 h at RT. The membranes were then washed 2 15 min with PBS and incubated in 1 : 5000 IRdye-conjugated secondary antibodies (Rockland) for 1 h at RT. Following a further 2 15 min PBS wash, membranes were scanned and analysed using Odyssey analysis system (Li-COR). Each experiment was repeated three times and statistical analyses were performed using GraphPad Prism 3.03.

Results

Expression pattern of DISC1 in the adult mouse brain

To obtain an overview of DISC1 expression in the adult mouse brain, paraffin-embedded sections from different regions of a mouse brain were analysed for DISC1-ir using a specific anti-DISC1 antibody (D27). This antibody has been described and characterized previously.¹² In order to demonstrate antibody specificity in mouse tissue, preabsorption with the immunizing peptide on the paraffin embedded sections from the same study was performed. The immunoreactivity seen in the cortex and hippocampus (Figure 1a, c) was clearly abolished by incubation of peptide with D27 antibody (Figure 1b, d).

Figure 1.

Digital photomicrographs of adult mouse brain showing specificity of polyclonal D27 antibody. (a, c) DISC1-ir (brown) in the cortex and hippocampus, respectively, on the paraffin-embedded sections of mouse brain. (b, d) Preabsorption of D27-ir with the immunizing peptide. Sections were counterstained with Mayers haematoxylin (blue). The immunoreactivity seen in the cortex and hippocampus (a, c) was clearly preabsorbed by the immunizing peptide (b, d).

Full figure and legend (127K)

Immunohistochemical analyses of different regions of adult mouse brain show that DISC1 protein is broadly expressed within several areas of the brain, especially in the cortex, hippocampus, hypothalamus, cerebellum and brain stem (Figure 2). In the cortex, expression was particularly high in layers II/III and V/VI (Figures 2a and 3a). However, isolated DISC1-ir cell bodies were scattered throughout the whole cortex. In the hippocampus, DISC1-ir was present in all regions, with the highest density in pyramidal cells of the CA3 region (Figures 2c, d and 3b, d). In the olfactory bulb (data not shown) and in the hypothalamus, DISC1-ir was intense throughout (Figure 2e, f). In the cerebellum, Purkinje cells were predominantly positive for DISC1 (Figure 2g, h). However, other small cells in the molecular layer and the granule layer also demonstrated some DISC1-ir (Figure 2h, arrows). In discrete regions of the brain stem, including spinal trigeminal nucleus (SP5N), lateral paragigantocellular nucleus (LPGi) and superior vestibular nucleus (SpVe), isolated neurons were particularly positive for DISC1 (Figure 2i, j).

Figure 2.

Digital photomicrographs of adult mouse brain showing the distribution of DISC1-ir (brown) in different brain regions: (a, b) in cortex, (c, d) in hippocampus, (e, f) in hypothalamus, (g, h) in cerebellum and (i, j) in brain stem. Photomicrographs in the right-hand panel (b, d, f, h, j) show enlarged view of boxes on the left-hand panel photomicrographs (a, c, e, g, i). Sections were counterstained with Mayers haematoxylin (blue). DISC1-ir of different density was observed in all studied areas of the brain. In the cortex (a, b) DISC1-ir was present in all layers with higher intensity in layers II–III, V–VI. In hippocampus (c, d), DISC1-ir was monitored in all regions with a higher intensity in CA3 regions. In hypothalamus (e, f), DISC1-ir was high through whole region. 3V—third ventricle, ARC—arcuate hypothalamic nucleus. In cerebellum, Purkinje cells (PCL) and also some other cells in the granule cell layer (GL) and molecular layer (ML) were DISC1-ir (arrows). In brain stem area (i, j), isolated neurons showed strong DISC1-ir. Sp5—spinal trigeminal tract; Sp5N—spinal trigeminal nucleus. Scale bars, 100 m (a, c, e, g, i), and 30 m (b, d, f, h, j).

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Figure 3.

Confocal images of adult mouse brain demonstrating DISC1-ir in neurons in different brain regions. (a) Colocalization of DISC1-ir (red) with MAP 2 (green) in the cortex, (b) hippocampus, (c) anterior hypothalamus. (d) Colocalization of DISC1-ir (red) with GFAP (green) in hippocampus. DISC1 expression colocalized with MAP2 (notice yellow colour in high power images in a–c). At the same time, DISC1 and GFAP did not demonstrate any coexpression (d). Scale bars, 50 m (a), 80 m (b–d), and 100 m (single labelling images in a–d).

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High power images (Figure 2, right-hand panel) reveal an uneven clustered pattern of DISC1 intracellular distribution in all studied regions of the brain. This pattern of DISC1 protein distribution is consistent with the punctate intracellular DISC1 distribution found in the primate and human brain (Oliver et al., unpublished observation) and also in various cell lines and primary neurons.^{12, 21, 28, 29}

DISC1 is expressed in neurons but not in astrocytes

In order to identify the type of cells in the adult mouse brain that express DISC1, frozen sections from adult mouse brain were processed for double immunofluorescent staining. DISC1 D27 antibody was used in combination with the neuronal marker MAP2, or astroglial marker GFAP. In different areas of the brain, DISC1 expression clearly colocalizes with MAP2 (Figure 3), suggesting a neuronal nature for DISC1-expressing cells. This is shown by the colocalization (yellow colour) of DISC1 in green and MAP2 in red in the cortex (Figure 3a), in the hippocampus (Figure 3b) and in the anterior hypothalamus (Figure 3c). Similar results were obtain for the olfactory bulb, cerebellum and brainstem (data not shown). Conversely, DISC1 and GFAP did not demonstrate any overlap in their expression anywhere in the brain as it clearly can be seen in the hippocampus (Figure 3d), suggesting that DISC1 is not expressed in glia.

We extended this study to identify the types of neurons expressing DISC1. Cortical brain sections were processed for double immunostaining with various neurochemical markers. Excitatory glutamatergic neurons were identified by using anti-glutamate transporter (EAAC1) antibody. -Aminobutyric acid (GABA)ergic interneurons were identified by applying anti-glutamic acid decarboxylase (GAD), anti-calbindin, anti-gephyrin and anti-calretinin antibodies. It is clear that in the mouse cortex, DISC1 colocalizes with glutamate transporter (EAAC1) positively expressing neurons (Figure 4a). DISC1-ir is also present in neurons expressing GAD (Figure 4c), gephyrin (Figure 4d) and the calcium-binding proteins calbindin (Figure 4b) and calreticulin (data not shown). These data suggest that in the cortex, DISC1 is expressed ubiquitously by both, excitatory and inhibitory neurons which might explain the potential multiple roles of DISC1. Further identification of possible subtypes of DISC1-positive neurons is ongoing.

Figure 4.

Confocal images of adult mouse cortical brain sections demonstrating colocalization of DISC1-ir with various neurochemical markers. (a) DISC1-ir (green) and EAACA-ir (red); (b) DISC1-ir (green) and calbindin-ir (red); (c) DISC1-ir (green) and GAD-ir (red); (d) DISC1-ir (green) and gephyrin-ir (red). DISC1-ir was present in all tested type of neurons, in neurons expressing EAAC1 (a), calbindin (b), GAD (c) and gephyrin (d) (notice yellow colour). Scale bars, 20 m (a–d).

Full figure and legend (639K)

Expression profile of DISC1 protein changes through development

To obtain an overview of DISC1 expression throughout mouse development, a quantitative Western blotting approach was used. Tissue lysates from mice of age E10.5 to E20.5 and from P1 to P35 were separated using SDS-PAGE, immunoblotted for DISC1 and then quantified by image analysis as described in Materials and methods. For embryonic samples, lysate from adult mouse cortex was used as a positive control. Using the antibody D27, two doublet bands were identified in all analysed samples. The low molecular weight doublet migrates at approximately 71 and 75 kDa and the larger molecular weight at approximately 100 and 105 kDa (Figures 5a and 6a). As discussed previously,^{12, 29} these bands most likely represent alternative splicing of the mouse DISC1 gene. Quantitation of immunoblots clearly demonstrate that the pattern of DISC1 expression changes during development, with the DISC1 isoforms having a unique profile. The 75 kDa isoform is already abundant at E10.5 and remains at a similar level through prenatal and postnatal developmental periods. The 70 kDa isoform is expressed at lower level during neonatal and early postnatal periods but is upregulated at P25 and remains high throughout 6 months of age, the latest time point that has been examined. The 105 kDa band is expressed at a low level at all time points, whereas interestingly, the 100 kDa band profile shows two significant peaks. The first peak is seen at E13.5 followed by a decrease by E16.5. The second peak occurs during postnatal development at P35 and then remains high through to 6 months of age (Figures 5a, c and 6a, c). To complement these data, the same lysates were immunoblotted for -tubulin. No changes through development in the expression profile of -tubulin were observed (Figures 5b and 6b).

Figure 5.

Distribution of DISC1 expression in the developing embryonic mouse brain. (a) Lysates from the pool of whole embryonic mouse brains at different time points (E10.5–E20.5) were blotted for D27 antibody to reveal DISC1-specific bands. Lysate from adult cortex was used as a positive control. (b) The same samples were blotted for -tubulin. (c) Quantification of immunoblots from three independent experiments are shown as four lines representing four different DISC1 bands, (d) or were plotted as meanSEM of band intensity (^*P<0.0175) measured using the Li-Cor Odyssey™. Two doublet bands were identified (71 and 75 kDa; 100 and 105 kDa) in all analysed samples (a). Quantitations of immunoblots demonstrated significant changes in the expression of the band 2 (100 kDa) during development with a peak at E13.5 (c, d).

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Figure 6.

Distribution of DISC1 expression in the postnatal mouse brain. (a) Lysates from the pool of a whole mouse brains at different time points (P1 to 6 months old) were blotted for D27 antibody to reveal DISC1-specific bands. (b) Same samples were blotted for -tubulin. (c) Quantification of immunoblots from three independent experiments are shown as four lines representing four different DISC1 bands, (d) or were plotted as meanSEM of band intensity (^**P<0.005) measured using the Li-Cor Odyssey™. Two doublet bands were again identified (71 and 75 kDa; 100 and 105 kDa) in all analysed postnatal samples (a). Quantitations of immunoblots demonstrated changes in the expression of the band 2 (100 kDa) during postnatal period with a significant increase at PD35 that remained high through to 6 months of age (c, d).

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Expression pattern of DISC1 in the embryonic mouse brain

To further explore the developmental pattern of DISC1 expression, immunohistochemistry of embryonic mouse brain tissue was performed. At an early embryonic age on days E 9.5–E10.5, the level of DISC1 immunoreactivity in the whole embryo was very low, almost negligible (Figure 7a). Later, at embryonic age E13.5–14, when cells start to migrate to the primary neuropallial cortex, DISC1-positive cells can be seen in several areas of the brain including the olfactory area, developing cortex, hypothalamus and pons (Figure 7b). At this stage, the cortical layer is very narrow but contains many DISC1-ir cells (Figure 7b, top inset). Remarkably, the olfactory nasal area has a very discreet number of isolated DISC1-positive cells (Figure 7b, bottom inset). At this time, DISC1-ir is also present in peripheral tissue such as heart and liver (data are not shown). At E17.5, when the brain displays an increased degree of differentiation, DISC1-ir is still strong in neopallial cortex, which is composed at this time of an inner broad nuclear layer (cortical plate) and a narrow outer layer (marginal zone). DISC1-positive staining can be seen in the intermediate zone of the wall of the lateral ventricle and midbrain area (Figure 7c). However, the strong DISC1-ir is no longer present in the olfactory area.

Figure 7.

Digital photomicrographs of embryonic mouse brain showing the distribution of DISC1-ir (brown) in the whole embryo at E10.5 (a) and in the embryo head at E14.5 (b) and E17.5 (c). Sections were counterstained with Mayers haematoxylin (blue). DISC1-ir can be seen in different areas, especially in developing cortex (neopallial cortex) and in developing hypothalamus (see high power images). DISC1 expression profile changed through early developmental stages from very low expression level at E10.5 (a) to high expression level in very discreet areas at E14.5 (b) and to adult like pattern of expression at E17.5 (c). Insets in (b) demonstrate a higher power illustration of developing cortex (top) and the olfactory area (bottom) at E14.5. Inset in (c) demonstrates a higher power illustration of developing cortex at E17.5. nc, neopallial cortex; h, hypothalamus; p, pons; oa, olfactory area; iz, intermidiate zone; mz, marginal zone; cp, cortical plate; sp, subplate. Scale bars, 20 m (a), 1 mm (b), 50 m (b, top inset), 30 m (b, bottom inset), 1 mm (c) and 70 m (c, inset).

Full figure and legend (361K)

It is clear that DISC1 expression has a very distinct profile through early mouse development.

Discussion

Schizophrenia is a neuropsychiatric disorder with a significant underlying genetic component and DISC1 has been identified as a potential risk gene based on several independent genetic studies.^{8, 9, 30, 31} The role and function of the DISC1 protein is currently under intensive investigation.^{12, 21, 22, 23, 28, 32} However, recent studies of DISC1 suggest a possible involvement in neurodevelopment. In the present study, we find further evidence for the role of DISC1 in brain development and, therefore, potential involvement in the neurodevelopmental abnormalities thought to contribute to schizophrenia.

For the first time, we describe the pattern of DISC1 protein distribution in the adult mouse brain. DISC1 is expressed in several brain regions, including olfactory bulb, cortex, hippocampus, hypothalamus, cerebellum and brain stem. Our results compliment and extend several other recent studies describing the DISC1 mRNA expression in mouse,³³ mRNA expression in rat^{21, 22, 34} and also mRNA and protein expression in primate.²³ However, there are some discrepancies between published data regarding the profile of DISC1 mRNA in the rodent brain. In the study by Ma et al.,³³ it has been suggested that in the mouse cerebellum mRNA signal can be detected in Bergmann glia, whereas in the findings by Honda et al.³⁴ DISC1 mRNA is expressed only in neurons through the whole rat brain including Purkinje cells of cerebellum. These differences could be due to the method sensitivity or variations in the experimental design. Results from the present study provide further evidence for the neuronal nature of DISC1 protein expression which clearly could be seen by colocalization with neuronal marker MAP2 and supports DISC1 mRNA profile by Honda et al.

Together, these findings suggest that the overall pattern of DISC1 expression is conserved across species. Interestingly, cortex and hippocampus are regions strongly implicated in the pathophysiology of schizophrenia.^{35, 36, 37, 38} It was previously reported that density of neurons can be significantly reduced in certain layers of the cortex of schizophrenics. For example, schizophrenic subjects showed a significant decrease in density of nonpyramidal neurons in layer II and pyramidal neurons in layer IV of the anterior cingulated cortex.³⁹ Another study demonstrated a reduced density of nonpyramidal neurons also in layers III, V and VI.⁴⁰ These findings suggest that the loss of certain types of neurons is a significant feature of schizophrenia. Therefore, expression of DISC1 in layers II/III and V/VI of the mouse cortex may indicate layers of neurons that may be more likely to be affected in schizophrenia, enhancing the possibility that DISC1 may contribute to the pathophysiology of the disorder.

Our results also demonstrate that DISC1 is specifically expressed in neurons, and appears to be largely absent from astroglia. This is in agreement with data from a number of recent studies. For example, DISC1 interacts with many neuronal proteins such as NUDEL and FEZ1. It is also involved in neurite outgrowth of PC12 cells.^{12, 21, 22} Furthermore, it is expressed in mouse primary neurons (Brandon et al., in preparation). Association of DISC1 with cytoskeletal elements^{21, 22, 28} together with findings that DISC1 is a part of a NUDEL-Lis1 complex¹² strongly suggests a role for DISC1 in neuronal migration. Indeed, the neuropathology of schizophrenia, which is based on observations of reduced brain mass and increased ventricular volume in schizophrenic subjects,³⁶ reflects abnormalities at the level of individual neurons. In spite of broad DISC1-ir in the brain, it is clear that it is not ubiquitously expressed by all neurons. However, we have not been able to identify a particular type of neurons that is DISC1 positive. Our results suggest that DISC1-expressing cells at least can be positive for both glutamate and GABA. It is well established that these two main neurotransmitters in the mammalian brain, glutamate and GABA, are implicated in the pathophysiology of schizophrenia.^{41, 42, 43, 44, 45, 46} The level of GABA itself is reduced in the hippocampus, and the number of markers for GABAergic neurons, such as calcium-binding proteins (CBPs) and GAD, are lost, particularly in cortical regions.^{41, 47, 48, 49} Increased binding to GABA-A receptors in schizophrenia has been reported in the frontal cortical regions⁵⁰ and hippocampus.⁵¹ Antagonists of NMDA receptors, phencyclidine (PCP) and ketamine can cause positive, negative and cognitive symptoms resembling schizophrenia in healthy individuals and can exacerbate psychotic symptoms in patients with schizophrenia.⁵² In this context, it will be of interest to further explore the role of DISC1 in the function of both GABAergic and glutamatergic neurons.

In agreement with previous reports^{21, 22} DISC1 exists as a number of isoforms including doublets of approximately 75 and 100 kDa, due to alternative splicing in the mouse DISC1 gene.^{21, 29} Critically though, all four DISC1 bands, within which there are two doublets, have a different expression profile in mouse brain through development, suggesting multiple roles for DISC1 in the CNS. Interestingly, only the expression of the 100 kDa band significantly changes through mouse developmental stages, firstly with upregulation at E13.5 when major developmental processes, such as neurogenesis and cellular migration in the brain, start to take place, and secondly, around P35 when the mouse is entering puberty. Remarkably, Nudel forms a complex predominantly with the 105 kDa band¹² which does not change dramatically during embryonic development but declines after birth. These results are in general agreement with work by Ozeki et al., suggesting upregulation for both DISC1 forms (75 and 100 kDa) during the embryonic period of rat development and then downregulation after birth. In future studies, it will be important to define protein partners of 100 kDa isoform, the most changeable isoform of DISC1. We are currently investigating potential interacting partners via application of yeast two-hybrid and mass spectrometry technology.

Immunocytochemical analyses of embryonic tissue strongly correlates with the pattern of DISC1 expression indicated by Western blot analyses. DISC1 immunoreactivity is very weak at E10.5, the period of development characterized by closure of the caudal neuropore.⁵³ At E13.5–14 when cells start to migrate to the primary neuropallial cortex, a significant increase in DISC1 expression in discrete areas such as olfactory area, cortex, hypothalamus and pons can be seen. At E17.5, DISC1-ir remains strong in neopallial cortex and is also present in the intermediate zone of the wall of the lateral ventricle and midbrain area. At the same time, DISC1-positive cells are no longer present in the olfactory area. These results raise the possibility that DISC1 may be an important factor in the developing mouse brain, especially required for normal cortical development. Previous post-mortem cytoarchitectural studies reported abnormal cortical and subcortical neuronal size, number and position in the brain of schizophrenic patients.⁵⁴ Structural in vivo neuroimaging studies describe a wide range of changes in cranial, cortical and subcortical structure in schizophrenic patients as compared to healthy controls.^{55, 56} In this context, it is of interest to speculate whether the DISC1 protein may be a potential biomarker in understanding the early pathological molecular mechanisms and events that culminate in schizophrenia.

Our findings that DISC1 expression is upregulated at puberty, a period corresponding to the clinical onset of schizophrenia,¹ once again reinforce a possible relevance of DISC1 to this disorder. Development of schizophrenia during the reproductive period suggests that the disorder is related to a disturbance in the balance between circulation of neuroexcitatory reproductive hormones to the brain and body and required compensatory changes in neurophysiology, and consequent remodeling of synapses in specific brain areas.¹ Receptors for and neurons containing reproductive hormones are strongly expressed in the hypothalamus, an area which is enriched with DISC1-ir neurons.

Taken together, our findings further expand our knowledge about the function of a recently described genetic risk factor in schizophrenia, DISC1. Based on the pattern of expression through development and in the adult mouse brain, we propose that DISC1 may play an important role in neurodevelopment, especially in corticogenesis, and may be an important factor and indicator for molecular events leading to the development and onset of schizophrenia.

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