Journal of Neuroscience Research 86:2190–2200 (2008)
Maternal Immune Activation in Mice Delays Myelination and Axonal Development in the Hippocampus of the Offspring Manabu Makinodan,1* Kouko Tatsumi,2 Takayuki Manabe,2 Takahira Yamauchi,1 Eri Makinodan,2 Hiroko Matsuyoshi,2 Shigero Shimoda,1 Yoshinobu Noriyama,1 Toshifumi Kishimoto,1 and Akio Wanaka2 1
Department of Psychiatry, Nara Medical University Medical School, Kashihara City, Nara, Japan Department of Anatomy and Neuroscience, Nara Medical University Medical School, Kashihara City, Nara, Japan 2
Epidemiological data suggest a relationship between maternal infection and a high incidence of schizophrenia in offspring. An animal model based on this hypothesis was made by injecting double-stranded RNA, polyinosinic-polycytidylic acid (poly-I:C), into early pregnant mice, and their offspring were examined for biochemical and histological abnormalities. Mouse brains were examined with special reference to oligodendrocytes, which have been implicated in several neurodevelopmental disorders. We detected a significant decrease of myelin basic protein (MBP) mRNA and protein at early postnatal periods in poly-I:C mice. MBP immunocytochemistry and electron microscopy revealed that the hippocampus of juvenile poly-I:C mice was less myelinated than in PBS mice, with no significant loss of oligodendrocytes. In addition, axonal diameters were significantly smaller in juvenile poly-I:C mice than in control mice. These abnormalities reverted to normal levels when the animals reached the adult stage. These findings suggest that retarded myelination and axonal abnormalities in early postnatal stages caused by maternal immune activation could be related to schizophreniarelated behaviors in adulthood. VC 2008 Wiley-Liss, Inc. Key words: oligodendrocyte; myelination; infection; schizophrenia; animal model
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There are multiple lines of evidence indicating myelination abnormalities in subjects with schizophrenia. Microarray data (Hakak et al., 2001; Tkachev et al., 2003; Sugai et al., 2004), white matter imaging techniques (Lim et al., 1999; Foong et al., 2000, 2002), and ultrastructural studies (Uranova et al., 2001) have shown the involvement of oligodendrocytes. The mechanisms behind this myelination abnormality and how it contributes to schizophrenic symptoms remain unknown. There are limitations to elucidating such mechanisms in human subjects; hence the need for animal models that can mimic schizophrenia, at least to some extent. ' 2008 Wiley-Liss, Inc.
So far, there have been various animal models of schizophrenia, such as the amphetamine model (Snyder, 1973), the phencyclidine model (Javitt and Zukin, 1991), and the ventral hippocampal lesion model (SamsDodd et al., 1997; Lipska et al., 2002). In the present study, we employed the maternal immune activation model, in which early-pregnant mice receive synthetic polyriboinosinic-polyribocytidilic acid (poly-I:C) injections and their pups are examined extensively for biochemical, morphological, and behavioral abnormalities (Zuckerman et al., 2003; Fortier et al., 2004; Meyer et al., 2006; Ozawa et al., 2006). The poly-I:C model is thought to impair the developmental processes of the brain, including glial differentiation, and is considered to be the least artificial model because it does not involve treatment with high drug doses. The implication of maternal immune activation in the etiology of schizophrenia has been underscored mainly by epidemiological studies. Maternal infection with the influenza virus (Shi et al., 2003; Brown et al., 2004a), polio virus (Suvisaari et al., 1999), herpes simplex virus type 2 (Buka et al., 2001), rubella (Brown et al., 2001), and toxoplasma (Brown et al., 2005) have been associated with a higher incidence of schizophrenia in the offspring. Elevated interleukin-8 in maternal serum is linked to a higher risk of schizophrenic offspring (Brown et al., 2004b). PolyI:C is commonly used to induce an immune response Contract grant sponsor: Ministry of Education, Science, Sports and Culture, Grant-in-aid for Scientific Research (C); Contract grant sponsor: Grant-in-aid for Young Scientists (B). *Correspondence to: Manabu Makinodan, MD, Department of Psychiatry, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara City, Nara 634-8521, Japan. E-mail:
[email protected] Received 25 October 2007; Revised 13 December 2007; Accepted 4 January 2008 Published online 25 April 2008 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.21673
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similar to that induced by viral infection (Zuckerman et al., 2003; Fortier et al., 2004; Guillot et al., 2005). Previous reports showed that poly-I:C mice have reduced prepulse inhibition, increased glial fibrillary acidic protein-positive cells, decreased expression of reelin, neuronal atrophy in the hippocampus, and reduced thickness of the hippocampus (Fatemi et al., 1999, 2002, 2004; Shi et al., 2003; Ozawa et al., 2006). However, hardly any oligodendrocyte abnormalities were reported in this model. The onset of maternal immune activation is at about E9.5 in this model (Shi et al., 2003), which coincides with the generation of Olig2-positive cells in the medial ganglionic eminence (MGE) of the murine telencephalon (Bansal et al., 2003). Injection of poly-I:C induces elevated levels of several cytokines, such as interleukin-1b, interleukin-6, inerleukin-10, and tumor necrosis factor (Fortier et al., 2004; Meyer et al., 2007; Smith et al., 2007), and some of these cytokines may affect the development and function of oligodendrocytes and could influence behavior offspring at a later stage. In this study, we demonstrated that maternal immune activation resulted in altered myelination and axonal development in the hippocampus of juvenile offspring. MATERIALS AND METHODS Animals and Prenatal Treatment C57BL/6 mice were mated at about 3 months of age, and the first day after copulation was defined as embryonic day 0 (E0). Pregnant mice received either a single intraperitoneal injection of poly-I:C (60 mg/kg) dissolved in phosphatebuffered saline (PBS), or an equivalent volume of PBS at embryonic day 9.5 (E9.5). Pups born from poly-I:C-treated mothers and PBS-treated mothers are hereafter referred to as poly-I:C mice and PBS mice, respectively. Pups were weaned and housed four or five per cage according to sex and litter at postnatal day 21 (P21) and housed in a temperature- and humidity-controlled animal facility under a reversed light-dark cycle (lights on 8:00–20:00). The prepulse inhibition of male pups from at least two different litters were measured at P63, and the other pups were sacrificed at P14 or P63 for immunohistochemistry and biochemical quantitative analyses. All animals were maintained with food and water ad lib throughout the experiments. Experimental protocols were according to the guidelines of the Animal Care Committee of Nara Medical University and were in accordance with the policies established in the NIH Guide for the care and use of laboratory animals. Prepulse Inhibition Test The mouse was placed in a translucent acrylic cage (7 cm 3 7 cm 3 16.5 cm). Movements of the animals were detected by a piezoelectric accelerometer (GH313A, GA245SO; Keyence, Kyoto, Japan) attached at the bottom of cage. White noise at 115 dB with duration of 50 msec was used as the acoustic startle stimulus (pulse). A noise prepulse of 85 dB was presented for 30 msec. Background noise was kept at a relatively constant level, 70–73 dB. The test session Journal of Neuroscience Research
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consisted of a total of 19 trials: 10 startle trials without a prepulse (habituation), followed by nine trials of prepulse test session. The mean intertrial interval was 25 sec (range 15–45 sec). In the prepulse trials, prepulse with lead time of 50 msec was followed by the pulse. Four pulse-alone trials and five prepulse trials were presented in random order. Relative startle response (RSR) was calculated using the formula RSR 5 PP/N, where PP is the mean response with prepulse and N is the mean response without a prepulse (n 5 13 poly-I:C mice, n 5 16 PBS mice). Real-Time RT-PCR and Western Blotting Brain tissue from at least two different litters of each group was dissected at P10, P14, or P63, and the hippocampus, including the fimbria hippocampi, and the cerebral cortex, including the corpus callosum, were removed carefully. Tissue was processed immediately for real-time RT-PCR analysis (n 5 6 each at P14, P63) or Western blot analysis (n 5 3 each at P14, P63). For RT-PCR, total RNA was isolated by using the Trizol extraction method (Life Technologies, Rockville, MD). cDNA was synthesized with Superscript-II Reverse Transcriptase (Invitrogen, Carlsbad, CA) and oligo-dT primer in accordance with the manufacturer’s protocols. Expression of related genes was quantified by using the Sybr Green reagent (23 Sybr Green Supermix; Bio-Rad, Hercules, CA) on a Bio-Rad iCycler, following the manufacturer’s instructions. PCR was performed under optimized conditions: 958C; denaturing, 558C; annealing, 728C; elongation, using the following primers: myelin basic protein (MBP); 384 bp, forward 50 GCGGGGCTCTGGCAAGGACTCACACACG-30 , reverse 50 -GCGGCTGTCTCTTCCTCCCCAGCTAAATC-30 ; 154 bp, forward 50 -GCGGGGCTCTGGCAAGGTACCCTGG-30 , reverse 50 -GCGGCTGTCTCTTCCTCCCCAGCTAAATC30 ; 169 bp, forward 50 -GCGGGGCTCTGGCAAGGTACCC TGG-30 , reverse 50 -GCCCCTCGGCCCCCCAGCTAAATC30 ; 195 bp, forward 50 -GCGGGGCTCTGGCAA GGTACCC TGG-30 , reverse 50 -GCGGCTGTCTCTCTTCCTCCCAGC TTAAAG-30 ; and GAPDH, forward 50 -AACTCCCTCAAGATTGTCAGCAA-30 , reverse 50 -GGCTAAGCAGTTGGT GGTGC-30 . No other products were amplified, because melting curves showed only one peak for each primer pair. Fluorescence signals were measured over 40 PCR cycles. The cycle number (Ct) at which the signals crossed a threshold set within the logarithmic phase was recorded. For quantitative analysis, the difference in cycle threshold (DCt) was evaluated between poly-I:C mice and PBS mice. The efficiency of amplification for each pair of primers was determined by serial dilutions of the templates. Each sample was normalized with GAPDH. Ct values used were the means of triplicate replicates. Experiments were repeated at least three times. For Western blotting, tissues were homogenized in a buffer containing 10 mM HEPES, 1 mM EDTA, 1 mM EGTA, 5 mM dithiothreitol, and 10 mM NaF. Sodium dodecyl sulfate (SDS) and 2-mercaptoethanol were added to the lysates, and samples were centrifuged at 12,000g for 10 min. Protein concentration of the supernatant was determined by BCA protein assay (Sigma, St. Louis, MO). Twenty micro-
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grams of total protein from whole-cell lysates was separated under reducing and denaturing conditions on a 12% SDSPAGE gel and electrotransferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). Nonspecific binding sites were blocked with 5% skim milk in PBS containing 0.05% Tween 20. The membranes were incubated with MBP antibody (rabbit polyclonal antibody; 1:1,000; Sigma), Olig2 antibody (rabbit monoclonal antibody; 1:1,000; Immuno-Biological Lab.), antiphosphorylated Akt antibody (specific to Ser 473 residue; rabbit monoclonal antibody; 1:1,000; Cell Signaling Technology, Beverly, MA), Akt (mouse monoclonal antibody; 1:1,000; Cell Signaling Technology) and b-actin antibody (mouse monoclonal antibody; 1:2,000; Sigma) for 4 hr at 378C. Membranes were then incubated in horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2,000) or antimouse IgG (1:2,000) for 12 hr at 48C, followed by ECL luminescence for detection of the antigens. Histology At P14 or P63, pups were anesthetized with pentobarbital and perfused with 4% paraformaldehyde in phosphate buffer. Brains were removed and postfixed in the same fixative at 48C. After overnight postfixation, brains were dehydrated in graded alcohols (70%; 1 hr, 80%; 2 hr, 95%; 2 hr, 100%; 4 3 2 hr) and xylene (2 3 1 hr). Subsequently, brains were blocked and embedded in paraffin. Sagittal sections were cut at 10 lm thickness and mounted on APS-coated slides. After overnight deparaffinization in fresh xylene, sections were rehydrated in graded alcohols to distilled water. After antigen activation with citric acid buffer and blocking in 5% bovine serum albumin (BSA), sections were first incubated overnight at 48C with antimyelin basic protein antibody (MBP; rabbit polyclonal IgG; 1:200; Sigma) or anti-Olig2 antibody (rabbit monoclonal antibody; 1:1,000; Immuno-Biological Lab.). Sections were then washed and incubated with biotin-conjugated anti-rabbit IgG (1:200; Vector, Burlingame, CA) for 1 hr at 378C. After washing, the sections were processed for 1 hr using a standard Vectastain ABC kit (Vector). Staining was visualized with diaminobenzidine (DAB; Vector) as a chromatic agent. Control slices were incubated as described above, without primary antibodies. No immunoreactivity was seen in controls (data not shown). Basic in situ hybridization procedures have been described previously (Mori et al., 2004). Briefly, paraffin-embedded sections of the above-mentioned time points were hybridized with digoxigenin (DIG)-labeled proteolipid protein (PLP) probe, which was detected by alkaline phosphatase-conjugated anti-DIG antibody using NBT/BCIP. PLP cDNA was amplified by PCR using primers 50 -AGAGAGAAA-30 and 50 -ATCAAAGAGAATATATTTG-30 , then cloned into pGEM3 vector. The sense or antisense PLP riboprobes were prepared by in vitro transcription with appropriate RNA polymerase in the presence of DIG RNA Labeling Mix (Roche, Indianapolis, IN). To investigate the histological characteristics in detail, the alveus of the hippocampal CA1 region at P14 and P63 and the layer VI of the frontal cortex at P14 were examined by electron microscopy. Briefly, after perfusion with 4% paraformaldehyde
(PFA), hippocampi were removed and postfixed with 2.5% glutaraldehyde for 2 hr at 48C. Then, they were immersed in 2% osmium tetraoxide for 1 hr, dehydrated in a graded series of alcohols, and embedded in a mixture of epoxy resin. To analyze quantitatively the thickness of the myelin sheath and axonal diameter, semithin sections (500 nm thick) were cut on an ultramicrotome (Ultracut-UCT; Leica, Japan) and stained with toluidine blue. Next, ultrathin sections were prepared (90 nm thick) for electron microscopy. These sections were stained with uranyl acetate and observed under the transmission electron microscope (1200EX; JEOL, Tokyo, Japan). Image Analyses The bands of Western blotting were analyzed with an image analysis program, ImageJ (http://rsb.info.nih.gov/ij/). The gross MBP-immunoreactive areas in three continuous, nonoverlapping images from arbitrary brain hemispheres were measured with ImageJ software, in the stratum lacnosummoleculare of CA1, in the pyramidal cell layer of the CA3 region of the hippocampus, and in the frontal cortex, by placing fixed squares in the various regions. Positive pixels of a region of interest were distinguished from negative ones by the automatic threshold process of the ImageJ program. The sum of the positive pixels (i.e., immunoreactive area) was averaged per animal (n 5 4 each at P14, P63). The number of Olig2 and PLP mRNA-positive cells within a fixed square box in the stratum lacnosum-moleculare of three continuous, nonoverlapping slices from arbitrary brain hemispheres was counted manually, and the individual mean values were calculated (Olig2: n 5 4 each, PLP mRNA: n 5 3 each). In stained semithin sections, light microscopic observation was performed at 31,000 magnification (Leica DMRXA), and the images of three continuous, nonoverlapping fiber cross-sections in the alveus of the CA1 region from the hippocampus from arbitrary brain hemispheres were acquired using a digital camera (FX380; Olympus) equipped with an image filing software (FLVFS-LS; Flovel). The number of axons was counted by each diameter, and the g-ratio was determined by dividing the circumference of an axon without the myelin by the circumference of the same axon including the myelin. More than 100 randomly chosen fibers per animal were analyzed by a researcher blind to the groups (n 5 5 each at P14, P63). Statistical Analysis The data are presented as mean 6 SEM. Statistically significant differences between groups were assessed by Student’s t-test. The v2 test was used to calculate the distribution of axon diameters. P < 0.05 was considered to be statistically significant.
RESULTS Postnatal Development of Poly-I:C Mice Appears To Be Similar to That of PBS Mice Before we compared the morphological and neurochemical characteristics of poly-I:C and PBS mice, the overall development was assessed by weighing the mice at P0 and P63. At birth, the body weight of the polyJournal of Neuroscience Research
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I:C mice (1.409 6 0.021 g, mean 6 SEM, n 5 24, consisting of three litters) was not significantly different from that of the PBS mice (1.426 6 0.011 g, mean 6 SEM, n 5 24, consisting of three litters; P > 0.05). At P63, both groups showed similar body weights (21.61 6 2.12 g for male poly-I:C mice from three litters, n 5 18, and 20.64 6 2.16 g for male PBS mice from three litters, n 5 18; P > 0.05). No visible abnormalities were observed for the poly-I:C or PBS mice throughout the postnatal period.
Decreased Prepulse Inhibition of Poly-I:C Mice in Adulthood The deficit of prepulse inhibition was noted in schizophrenic patients and many animal studies that have been undertaken to model the pathology of schizophrenia. In the present study, we measured the extent of prepulse inhibition of poly-I:C mice at P63 and compared it with that of PBS mice. The mean percentage of prepulse inhibition of poly-I:C mice was significantly decreased compared with that of PBS mice (Fig. 1; P < 0.05).
Fig. 1. Mean percentage of prepulse inhibition by auditory prepulse with 50 msec of lead time. The extent of prepulse inhibition of poly-I:C mice was decreased compared with that of PBS mice. Data are given as mean 6 SEM. The sample number is 13 poly-I:C mice and 16 PBS mice.
Myelination in the Poly-I:C mouse Hippocampus Is Impaired in an Early Postnatal Period We next surveyed oligodendrocyte abnormalities in poly-I:C mice of different postnatal ages and compared them with control PBS mice. Real-time RT-PCR revealed that all splicing variants of MBP mRNA (sizes of the amplified fragments were 585 bp, 507 bp, 462 bp, and 384 bp, corresponding to the 21.5-, 18.5-, 17-, and 14-kDa forms of the MBP protein, respectively) were significantly decreased in the P14 hippocampus (Fig. 2A; P < 0.05) of poly-I:C mice compared with PBS mice. This difference was no longer significant at P63 (Fig. 2C; P > 0.05). On the other hand, splicing variants of MBP mRNA in the cerebral cortex showed comparable levels in the poly-I:C and PBS mice, both at P14 and at P63 (Fig. 2B,D; cerebral cortex at P14; P > 0.05, cerebral cortex at P63; P > 0.05). GAPDH levels of all samples were comparable. MBP levels were examined by Western blotting. As expected from the mRNA data, MBP expression was decreased in the hippocampus of poly-I:C mice at P14 but not at P63. Furthermore, in
Fig. 2. Real-time RT-PCR data for MBP mRNA normalized to GAPDH. All splicing variants of MBP are decreased at P14 (A), but not at P63 (C) in the hippocampi of poly-I:C mice compared with PBS mice. In contrast, there is no difference in the expression of MBP in the cerebral cortex between poly-I:C mice and PBS mice at P14 (B) and P63 (D). Data are given as mean 6 SEM. The sample number of each group is six. Journal of Neuroscience Research
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Fig. 3. The Western blotting results for MBP protein represented as images (A) and densitometries (a). MBP proteins corresponding to all splicing variants are decreased at P14 but not at P63 in the hippocampus of poly-I:C mice compared with PBS mice. In contrast, there is no difference in the expression of MBP in the cerebral cortex between poly-I:C mice and PBS mice at P14 or P63. The expression of Olig2 in the hippocampus is not different between poly-I:C mice and PBS mice (B,b). Data are given as mean 6 SEM. The sample number of each group is three.
the cerebral cortex, there were no differences in MBP expression levels between poly-I:C mice and PBS mice at P14 or P63. (Fig. 3A,a; hippocampus P14, P < 0.05; hippocampus P63, P > 0.05; cerebral cortex P14, P > 0.05; cerebral cortex P63, P > 0.05). Olig2 is a transcription factor for oligodendrocyte development and is expressed in oligodendrocyte precursor cells and maturing and matured oligodendrocytes. At P14, Olig2 expression was not different between the two groups (Fig. 3B,b; P > 0.05). Having confirmed the above-described results, we next performed MBP immunostaining to study myelination in the hippocampal formation. MBP immunoreactivity was decreased in the stratum lacnosum-moleculare of the dorsal CA1 region and in the pyramidal cell layer of both the dorsal and the ventral CA3 region of polyI:C mice at P14 compared with PBS mice (Fig. 4A– F,K–N). No differences were found in the P14 cerebral
cortex (Fig. 4O,P). These results were confirmed with semiquantitative analysis in ImageJ software (Fig. 4a: dorsal hippocampus, P < 0.05; Fig. 4c: ventral hippocampus, P < 0.05; Fig. 4d: cerebral cortex, P > 0.05). On the other hand, MBP immunostaining at P63 revealed no difference in the hippocampal subregions or in the cerebral cortex (data not shown). Overall, poly-I:C mice at early postnatal time points suffered from delayed myelination in the hippocampus, but not in the cerebral cortex, and myelination levels reverted to control levels at later (young adult) stages. The delayed myelination suggests impaired oligodendrocyte development, which led us to examine the number and distribution of PLP mRNA-positive cells by in situ hybridization and Olig2-positive cells by immunohistochemistry. There was no significant difference in the number or distribution pattern of PLP mRNA-positive cells and Olig2-immunopositive cells Journal of Neuroscience Research
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Fig. 4. MBP immunoreactivity in the dorsal hippocampus (B) and ventral hippocampus (L) of poly-I:C mice is significantly decreased in the pyramidal cell layer of the dorsal CA3 (D) and ventral CA3 region (N) and in the stratum lacnosum-moleculare of the dorsal CA1 region (F) but not in the frontal cortex (P) at P14 compared with PBS mice
(A,C,E,K,M,O). There is no difference in the number of PLP-positive cells and Olig2-positive cells between the groups (G–J). The densitometries are shown next to the images (a–d). Data are given as mean 6 SEM (*P < 0.05). The sample number of each group was four, except the number for PLP was three. Scale bars 5 200 lm in L; 100 lm in P.
between poly-I:C mice and PBS mice at P14, despite the delayed myelination (Fig. 4G–J,b; P > 0.05). The number and distribution of neurons was not different between the two groups as determined by Nissl staining (data not shown). To investigate the altered myelination further, we analyzed axon diameters and g-ratios in semithin sections and studied axons with myelin sheath by electron microscope. Electron microscopic examination revealed the smaller axon diameter and thinner myelin sheath in the alveus of the hippocampal CA1 region of poly-I:C mice than of PBS mice at P14 (Fig. 5A,B), but such differences were no longer detected at P63 (Fig. 5C,D). In contrast to the hippocampus, layer VI of the frontal cortex at P14 showed a comparable axon diameter and myelin thickness between the two groups (Fig. 5E,F). Image analysis showed that the distribution of axon diameters in the alveus of CA1 between poly-I:C mice and PBS mice was different; poly-I:C mice had more small-diameter axons and fewer large-diameter axons than PBS
mice. Accordingly, the mean axon diameter of poly-I:C mice was significantly smaller than that of PBS mice at P14 (Fig. 5G,H; P < 0.05, respectively). Furthermore, the g-ratio was significantly increased in poly-I:C mice compared with PBS mice at P14 (Fig. 5I; P < 0.05). Taken together, these results indicate that the axon diameter is smaller and the myelin sheath thinner in polyI:C mice compared with PBS mice at P14. In contrast, the distribution of axon diameter, the mean axon diameter, and the g-ratio in the alveus of the CA1 region was not different between poly-I:C mice and PBS mice at P63 (Fig. 5J–L; P > 0.05, respectively).
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Decreased Akt Phosphorylation in the Hippocampus of Poly-I:C Mice The phosphatidylinositol/Akt pathway is thought to be involved in the proliferation and maturation of oligodendrocytes (Ebner et al., 2000; Flores et al., 2000) through axon–glial interactions. We hypothesized that
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Fig. 5. Electron microscopic images show that the axon diameter is small and the myelin sheath is thin in the alveus of the hippocampal CA1 region of poly-I:C mice (B) compared with PBS mice (A) at P14. However, no apparent differences were detected at P63 (C,D). There were also no differences in the axon diameter and myelin thickness in the layer VI of the frontal cortex at P14 between the two groups (E,F). Different axon diameter distribution patterns of poly-I:C mice and PBS mice (G; P < 0.05) and a decrease of the
mean axon diameter in poly-I:C mice (H; *P < 0.05) compared with PBS mice in the alveus at P14 in stained semithin sections. Increase in the g-ratio in the alveus in poly-I:C mice was detected compared with PBS mice (I; *P < 0.05). No difference in distribution of axon diameters, mean axon diameter, and g-ratio between poly-I:C mice and PBS mice at P63 (J–L, respectively; P > 0.05). The sample number of each group is four. Scale bar 5 1 lm.
this pathway may be attenuated in the poly-I:C mice and thereby cause delayed myelination. Western blotting experiments revealed that phosphorylation of Akt was decreased in the hippocampus of poly-I:C mice compared with PBS mice at P10, but not Akt protein levels (Fig. 6A; p-Akt: P < 0.05, Akt: P > 0.05). In contrast to the expression in the hippocampus at P10, the phosphorylation of Akt in the cerebral cortex at P10 was not different between poly-I:C mice and PBS mice (Fig. 6B; p-
Akt: P > 0.05; Akt: P > 0.05), and there were no differences in Akt phosphorylation in the hippocampus and the cerebral cortex between poly-I:C mice and PBS mice at p63 (Fig. 6C,D; p-Akt: P > 0.05; Akt: P > 0.05). DISCUSSION Increasing evidence from neuropathological and imaging studies supports the hypothesis that schizophreJournal of Neuroscience Research
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Fig. 6. There is a significant decrease of phosphorylated (p)-Akt in the hippocampus of poly-I:C mice compared with PBS mice at P10 hippocampus, whereas the amount of Akt is not changed (A). There were no differences in the expression of p-Akt and Akt in the cerebral cortex at P10 (B) or P63 (D) and in the hippocampus at P63 (C). Densitometry is shown next to the image. Data are given as mean 6 SEM. The sample number of each group is three.
nia is a neurodevelopmental disorder (Weinberger, 1987, 1995; Murray et al., 1992; Pearce, 2001; Meyer et al., 2005). Maternal immune activation and resultant abnormal behavior in the offspring are considered to be a typical model for such a neurodevelopmental hypothesis. Several reports have shown decreased prepulse inhibition, exploratory behavior, social behavior, and latent inhibition and increased sensitivity to dopamine agonists in poly-I:C mice, and the impairments of prepulse inhibition, latent inhibition, and cognitive function were improved with antipsychotics (Shi et al., 2003; Zuckerman and Weiner, 2005; Ozawa et al., 2006). Then, the abnormal behaviors do not become apparent until adulthood (Ozawa et al., 2006). In these reports, poly-I:C was injected intravenously in pregnant females at 4.0 mg/kg (rats) at E15 (Zuckerman et al., 2003), 20 mg/kg (mice) intraperitoneally at E9.5 (Shi et al., 2003), and 5.0 mg/kg (mice) at E12–E17 (Ozawa et al., 2006). Although we used a high dose of poly-I:C (60 mg/kg) at E9.5 in mice, mean body weight of the poly-I:C mice was not different from that of PBS mice at P0 and Journal of Neuroscience Research
P63. The poly-I:C mice appeared to be indistinguishable from control PBS mice regarding movement and feeding behavior (data not shown). Additionally, comparable activity in the open-field test, comparable spatial memory in the water-maze test, and decreased prepulse inhibition were detected in these poly-I:C mice at P63. Overall, poly-I:C mice in the present study did not suffer profoundly from retardation in their general development and would be suitable for the analysis of psychotic diseases such as schizophrenia. In our search for causes of abnormal behavior, such as a decreased prepulse inhibition, we focused on oligodendrocyte abnormalities. Many reports have shown evidence for an involvement of oligodendrocytes and abnormal myelination in the pathophysiology of schizophrenia (Hakak et al., 2001; Tkachev et al., 2003). Most studies have investigated oligodendrocytes and myelination in the prefrontal cortex. However, the hippocampus has also been studied as a brain region that may contribute to the induction of schizophrenia-like symptoms for the following reasons. 1) Hippocampal abnormalities
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have been given as a primary cause of schizophrenia (Bogerts et al., 1985; Arnold, 1999; Velakoulis et al., 1999; Weinberger, 1999). 2) Hippocampal abnormalities have been reported not only for subjects with schizophrenia but also for their parents and siblings (Seidman et al., 1999; Harris et al., 2002) and subjects with a schizotypal personality disorder showing preclinical symptoms before the onset of schizophrenia (Suzuki et al., 2005). 3) The CA1 region of the hippocampus is sensitive to various stresses, such as methylazoxymethanol injection in utero (Calcagnotto et al., 2002). 4) A neonatal excitotoxic lesion of the rat ventral hippocampus results in schizophrenia-like behavior (Chambers et al., 1996; Sams-Dodd et al., 1997; Lipska et al., 2002). In addition to these lines of evidence, recent reports revealed altered myelin-related gene expression in the hippocampus of schizophrenic patients (Katsel et al., 2005; Haroutunian et al., 2006). The present study revealed abnormalities in myelin formation in the hippocampus of poly-I:C mice both histologically and biochemically. No differences were found in the cerebral cortex, including prefrontal regions. MBP expression (mRNA and protein) was decreased from P14, but MBP expression had recovered to normal levels at P63. Immunohistochemical data confirmed the biochemical findings, and electron microscopy confirmed that myelination was retarded in poly-I:C mice. It is of note that no significant loss of oligodendrocytes was detected in this experimental model, insofar as the number of the PLP mRNA- and Olig2-immunopositive cells in the poly-I:C mice was comparable to that in control mice. Telencephalic oligodendrocyte progenitors originate from ventral forebrain at about E9.5 (He et al., 2001; Ivanova et al., 2003), the period coinciding with that of maternal immune activation. Along with the fact that myelination in the hippocampus of poly-I:C mice gradually reached normal levels by adulthood, immature oligodendrocytes (Olig21, PLP mRNA1) that were able to migrate to the hippocampus may stop transiently differentiating to myelinating oligodendrocytes by an unknown mechanism. Myelination in the murine brain is at its peak from P10 to P21 (Lu et al., 2005). This period is also critical for the plasticity of the neural circuit, e.g., the corticolimbic system (Lipska et al., 2002). Recent reports revealed that myelin and NogoR alter synaptic plasticity during this critical period (McGee et al., 2005), and neonatal ventral hippocampal lesion leads to excessive firing of pyramidal neurons in the prefrontal cortex in response to mesocortical stimulation in adult rats, but not before adulthood (O’Donnell et al., 2002). By analogy to the ventral hippocampal lesion model, it is possible that the hypomyelination in the juvenile hippocampus of the poly-I:C mice, which would result in reduced neural transmission in the hippocampus or in the corticolimbic system, might produce the altered firing in the prefrontal cortex and abnormal behaviors later in adulthood. It should, however, be emphasized that the recovery of myelination in adulthood in this model is different
from the situations in schizophrenic patients; oligodendrocyte abnormalities in schizophrenia are reported mostly for adult and not for juvenile patients (Davis et al., 2003; Tkachev et al., 2003; Chambers and Perrone-Bizzozero, 2004). One possible explanation for the discrepancy is that psychosocial stresses and/or high dose of antipsychotics may affect vulnerable oligodendrocytes in adult schizophrenic patients. Taking this discrepancy into account, we must be cautious in extrapolating the pathophysiology of schizophrenia from the present results. The role of oligodendrocytes in myelination has been discussed, but axonal factors are also necessary for myelination (Brophy, 2004; Sherman and Brophy, 2005). A previous report showed pyknosis of neurons in the hippocampal CA1 region in poly-I:C rats and disarray of the CA1–CA2 junction in influenza virus-infected mice, which is an alternative model for schizophrenia (Cotter et al., 1995; Zuckerman et al., 2003). In the present study, a mild but significant delay of axonal development in the hippocampus was detected in poly-I:C mice. It is possible that the occurrence of immature axons represents primary changes in this experimental paradigm and that hypomyelination is caused by a relative paucity of axonal factors such as neuregulin 1. It remains unknown how this delayed myelination and delayed axonal development in the juvenile period results in abnormal behavior in adulthood. There is a significant time lag between histological changes and behavioral abnormalities, and putative links between these two events could become key factors amenable to therapeutic intervention. In this context, a recent report provided an interesting observation; conditional expression of dominant negative erbB4, which is a major receptor for neuregulin 1, in postnatal mice resulted in alteration of oligodendrocyte morphology, dopaminergic hyperactivity, and neuropsychiatric behavior (Roy et al., 2007). Phosphorylated Akt, a downstream factor of the erbB signaling pathway, was decreased in P10 poly-I:C hippocampus (Fig. 6). Axon–glial interactions, including neuregulin 1-erbB signaling, might thus be altered in the juvenile stage, and this could cause dopaminergic signaling abnormalities in the poly-I:C mice. We believe that this issue should be addressed and that further investigations are needed to understand the complex nature of schizophrenia. ACKNOWLEDGMENTS We thank Ms. Ryoko Sakumura for her excellent assistance with the histology experiments. REFERENCES Arnold OH. 1999. Schizophrenia—a disturbance of signal interaction between the entorhinal cortex and the dentate gyrus? The contribution of experimental dibenamine psychosis to the pathogenesis of schizophrenia: a hypothesis. Neuropsychobiology 40:21–32. Bansal R, Lakhina V, Remedios R, Tole S. 2003. Expression of FGF receptors 1, 2, 3 in the embryonic and postnatal mouse brain compared with Pdgfralpha, Olig2 and Plp/dm20: implications for oligodendrocyte development. Dev Neurosci 25:83–95. Journal of Neuroscience Research
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