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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Stress and electroconvulsive seizure differentially alter GPR56 expression in the adult rat brain Go Suzukia,1 , Yasunari Kandab,1 , Masashi Nibuyaa,⁎, Takeshi Hiramotob , Teppei Tanakaa , Kunio Shimizuc , Yasuhiro Watanabeb , Soichiro Nomuraa a
Department of Psychiatry, National Defense Medical College, Namiki 3-2, Tokorozawa, Saitama 359-8513, Japan Department of Pharmacology, National Defense Medical College, Japan c Division of Behavioral Science, National Defense Medical College Research Institute, Japan b
A R T I C LE I N FO
AB S T R A C T
GPR56, a member of the G-protein-coupled receptor family, plays a role in the formation of the
Accepted 4 September 2007
frontal and parietal brain lobes and cortical lamination in the embryonic stage. A recent report
Available online 20 September 2007
indicated the existence of GPR56 transcripts in the subventricular zone (SVZ) and hippocampal subgranular zone (SGZ) of the adult mouse brain. Both these regions are known to continually
produce neural progenitor cells in the adult brain. Here, we demonstrate abundant GPR56
protein expression in the ependymal cell layer and SVZ as well as its reciprocal translational
Ependymal cell layer
regulation by a 12-day behavioral stress paradigm and 10-day electroconvulsive seizure (ECS)
treatment. Our study revealed that GPR56 transcript expression in the hippocampus was
Brain-derived neurotrophic factor
regulated by stress and seizure in a manner identical to that in the SVZ. GPR56 expression was
downregulated by stress and upregulated by the ECS treatment in both regions, whereas nestin
expression showed no changes. Western blot analysis revealed a robust ECS-induced increase in brain-derived neurotrophic factor expression in the wall of the lateral ventricle including the ependymal cell layer and the SVZ, which may provide a possible regulatory mechanism for GPR56 expression. We consider that GPR56 is expressed in the ependymal cell layer and in immature progenitor cells and that its expression is regulated by functional stimulation. © 2007 Elsevier B.V. All rights reserved.
The involvement of GPR56, a member of the secretin receptorlike G-protein-coupled receptor family, in brain development has been shown. GPR56 is homologous to the 7 transmembranedomain receptor superfamily and possesses a long extracellular N-terminus that is considered to be related to cell adhesion and
intracellular signaling (Liu et al., 1999; Stacey et al., 2000). Mutations at the N-terminus of GPR56 cause a brain cortical malformation called bilateral frontoparietal polymicrogyria in humans (Piao et al., 2004). In this condition, patients are mentally retarded and have abnormally numerous small gyri with disorganized cortical lamination, particularly in the frontal cortex. In addition to abundant neuronal expression during the
⁎ Corresponding author. Fax: +81 42 996 5203. E-mail address: [email protected]
(M. Nibuya). Abbreviations: BDNF, brain-derived neurotrophic factor; BrdU, bromodeoxyuridine; CC, corpus callosum; CPu, caudate putamen; CVS, chronic variable stress; DG, dentate gyrus; ECS, electroconvulsive seizure; FBS, fetal bovine serum; GFAP, glial fibrillary acidic protein; HRP, horseradish peroxidase; LSN, lateral septal nucleus; LV, lateral ventricle; NeuN, neuronal-specific nuclear protein; OB, olfactory bulb; PSANCAM, polysialic acid-neural cell adhesion molecule; PTSD, post-traumatic stress disorder; SGZ, subgranular zone; SVZ, subventricular zone 1 S.G. and K.Y. contributed equally to this work. 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.09.020
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embryonic period, preferential expression of GPR56 mRNA has been shown in neuronal progenitor cells in the subventricular zone (SVZ) around the lateral ventricle (LV) and in the hippocampal subgranular zone (SGZ) of the adult mouse brain (Piao et al., 2004). The exact expression pattern of cells in the SVZ that express the GPR56 protein and its transcriptional and translational regulation have not yet been elucidated. The downregulation of GPR56 expression in a renal cell carcinoma cell line due to hypoxia has been demonstrated (Maina et al., 2005). Moreover, the activation of the cytoskeletal signaling and adhesion process by overexpression of GPR56 in a glioma cell line has been reported (Shashidhar et al., 2005). Although further investigations are still required, it is believed that the translational regulation of GPR56 protein in tumor cells is related to the metastatic process of carcinomas (Xu and Hynes, 2007). Neural progenitors generated in the SGZ migrate and differentiate into mature neurons in the granule cell layer of the hippocampal dentate gyrus (DG) (van Praag et al., 2002). Likewise, neuroblasts in the SVZ migrate through the rostral migratory stream toward the olfactory bulb (OB) and differentiate into neurons in the granular and periglomerular cell layers (Doetsch and Alvarez-Buylla, 1996). It has been reported that newly generated neurons in both DG (Madsen et al., 2003) and OB (Gheusi et al., 2000) are functionally integrated and active in the adult mammalian brain. The hippocampus and OB are known to play important roles in memory and learning in rodents (Stevens, 1996; Magavi et al., 2005). In addition, it is believed that the hippocampus and OB are related to emotional behavior, and increased aggression and anxiety have been demonstrated in neural cell adhesion molecule knockout mice with disturbed postnatal development in both regions (Stork et al., 2000). Although many investigations have reported reduced neurogenesis in the hippocampal SGZ after behavioral stress loading (Gould and Tanapat, 1999; Warner-Schmidt and Duman, 2006), only a small number of studies have examined the effects of stress on neural proliferation in the SVZ, and their results are still controversial. It has been shown that chronic social defeat stress and repeated inescapable electric foot shocks did not alter the proliferation rate in the SVZ as determined by bromodeoxyuridine (BrdU) labeling (Czeh et al., 2007; Chen et al., 2006). Moreover, chronic restraint stress treatment did not influence the number of progenitor cells detected by Ki67 immunoreactivity (Kaneko et al., 2006a). On the other hand, it has been reported that animals with streptozotocin-induced diabetes mellitus showed stress vulnerability (Hirano et al., 2006), and another study demonstrated reduced cell proliferation in both SVZ and SGZ of streptozotocin-induced chronic diabetic mice as deter-
mined by BrdU labeling (Saravia et al., 2004). Increased proliferation of neural precursor cells in the adult rodent SVZ (Parent et al., 2002) and SGZ (Parent et al., 1997) due to various seizure paradigms has been reported. Currently, investigators are interested in whether decreased neurogenesis is involved in the process of hippocampal atrophy in patients with posttraumatic stress disorder (PTSD) and depression (Sapolsky, 2000) and whether increased neurogenesis is involved in the process of dispersal and widening of the granule cell layer of the hippocampal DG in epilepsy patients (Houser, 1990; Jessberger et al., 2005). In the present study, we attempted to elucidate the effects of stress and electroconvulsive seizure (ECS) treatments on GPR56 expression in the rat hippocampus and SVZ. In the hippocampal region, the correlation of several mechanisms including the hypothalamic–pituitary–adrenal axis (Brown et al., 1999; Mirescu et al., 2004), neurotrophin pathway (Duman and Monteggia, 2006) and inflammatory/immunological sequel (Monje et al., 2003; Kaneko et al., 2006b) with a stress-induced decrease in neurogenesis has been implicated. A recent report indicated that chronic cholinergic stimulation commonly promotes the survival of newborn neurons in both DG and OB, but does not affect the proliferation of progenitor cells in the SVZ and SGZ (Kaneko et al., 2006a). A neurotrophic and protective effect of estrogen, which restores reduced neurogenesis in the SVZ and SGZ, has been reported in streptozotocin-induced diabetic mice (Saravia et al., 2004). Besides these observations, little is known about the common physiologic components that influence neurogenic activity in both SVZ and SGZ. Therefore, we also examined the change in brain-derived neurotrophic factor (BDNF) expression in the LV wall including the ependymal cell layer and the SVZ adjacent to the caudate putamen (CPu); this factor might be involved in regulating the proliferation and differentiation of neural progenitor cells. BDNF is known to be robustly induced by ECS treatment in both hippocampus and frontal brain regions (Nibuya et al., 1995).
2.1. Expression of GPR56 immunoreactivity around the lateral ventricle (LV) Immunohistochemical examinations revealed restricted GPR56 expression in the adult rat brain. GPR56 immunoreactivity was most prominent around the LV, as shown in Fig. 1A. This immunoreactivity was not detected in the hippocampal region when the presently available polyclonal antibody was
Fig. 1 – [A] Immunostaining for GPR56 around a rat lateral ventricle (LV) section adjacent to the caudate putamen (CPu), lateral septal nucleus (LSN) and corpus callosum (CC). [B1a, 2a, 3a, 4a and 5a] The ventral LV portion, represented with a gray rectangle in panel A, is immunostained for GPR56 (green). [B1b, 2b, 3b, 4b and 5b] The same section is also immunostained for NeuN (red), GFAP (red), Ki67 (red), PSA-NCAM (red) and nestin (red). [B1c, 2c, 3c, 4c and 5c] The two images shown on the left were superimposed. In the ventral portion, GPR56 immunoreactivity overlapped the nestin immunoreactivity in the ependymal cell layer and subventricular zone (SVZ), but no co-localizations were observed with NeuN-, GFAP-, Ki67- and PSA-NCAM-positive cells. [C1a, 2a, 3a and 4a] The dorsal LV portion, represented by a yellow rectangle in panel A, is immunostained for GPR56. [C1b, 2b, 3b and 4b] The same section is also immunostained for NeuN, GFAP, Ki67 and PSA-NCAM. [C1c, 2c, 3c, and 4c] The two images shown on the left were superimposed. GPR56 immunoreactivity exhibited no co-localizations with NeuN-, GFAP-, Ki67- and PSA-NCAM-positive cells. [D] The magnified view of the dorsal LV portion adjacent to the CPu is represented by a blue rectangle in panel A. GPR56 immunoreactivity is restricted to a line of the ependymal cell layer in this region.
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Fig. 2 – [A1a, 1b and 1c] In the dorsal LV portion adjacent to the caudate putamen (CPu), GPR56 immunoreactivity did not coincide with nestin-positive cells. [B1a, 1b and 1c] In this region, GPR56 was expressed only in the ependymal cell layer and not in the nestin-positive cells in the subventricular zone (SVZ). [A2a, 2b, 2c, B2a, 2b and 2c] The nestin-positive cells in this region did not exhibit GFAP immunoreactivity. [A3a, A3b, A3c, B3a, B3b and B3c] The nuclei of many nestin-positive cells in the SVZ were Ki67-positive; these cells were considered to be neuroblasts. The yellow and blue rectangles correspond to the same regions indicated in Fig. 1A.
used in the present immunohistochemical detection method (data not shown). In the ventral LV portion, represented with a gray rectangle in Fig. 1A, GPR56 immunoreactivity completely coincided with nestin-expressing cells in the ependymal cell layer and SVZ (Fig. 1B5). GPR56 was not co-expressed with neuronal-specific nuclear protein (NeuN; Fig. 1B1) and glial fibrillary acidic protein (GFAP; Fig. 1B2), which are considered to be expressed in mature neurons and glial cells. In addition, GPR56 immunoreactivity was not co-expressed with Ki67 (Fig. 1B3) and polysialic acid-neural cell adhesion molecule (PSA-NCAM; Fig. 1B4), which are considered to be expressed in neuroblasts. In the dorsal LV, GPR56 immunoreactivity did not coincide with nestin-expressing cells (Fig. 2A1). In the dorsal LV adjacent to the CPu, GPR56 was expressed in the line of ependymal cell layer but not in the nestin-positive cells of the SVZ (Fig. 2B1). Moreover, in this region, GPR56 immunoreactivity did not coincide with NeuN (Figs. 1C1 and D1), GFAP (Figs. 1C2 and D2), Ki67 (Figs. 1C3 and D3) or PSA-NCAM (Figs. 1C4 and D4) immunoreactivities. In the dorsal SVZ region adjacent to the CPu, many nestin-positive cells co-expressed Ki67 (Figs. 2A3 and B3) without expressing GFAP immunoreactivity (Figs. 2A2 and B2);
these cells were considered to be dividing neuroblasts, but they did not express GPR56 immunoreactivity. The GPR56 polyclonal antibody used in this study was immunized with the polypeptide of the N-terminal extracellular domain. The immunoblot revealed an appropriate molecular weight band of the protein, and increased expression of this protein has been demonstrated in glioblastoma and astrocytoma tissues by immunocytochemical procedures (http://www.mblintl.com/mbli/index.asp). The specificity of the antibody, however, has not been demonstrated by using the GPR56-knockout tissues. All immunostainings were performed using 4 to 6 different rats, and the same immunoreactivity pattern was obtained in each individual rat.
2.2. Effects of chronic variable stress (CVS) and ECS on GPR56 expression and the nestin protein in the LV wall adjacent to the CPu Western blotting revealed that CVS treatment significantly reduced GPR56 expression in the LV wall including the ependymal cell layer and the SVZ adjacent to the CPu (Fig. 3B). The amount of GPR56 protein was 13%± 2% in the CVS-treated group (n =6) compared to 100%± 28% in the control group (n =6), after
Fig. 3 – Western blot analysis of protein expression in the lateral ventricle (LV) wall including the ependymal cell layer and the SVZ. [A and C] GPR56 protein expression was significantly enhanced by electroconvulsive seizure (ECS) treatment. [B and C] GPR56 protein expression was significantly decreased by chronic variable stress (CVS) treatment. [D, E and F] The expression of 400-kDa nestin protein was not influenced by the ECS and CVS treatments. [G and H] The effect of ECS in this region was confirmed by the enhanced expression of brain-derived neurotrophic factor (BDNF). During electrophoresis, 20 μg protein [A, D, E and G] or 40 μg protein [B] of the homogenates from the rat LV wall adjacent to the caudate putamen (CPu) was applied in each lane. The relative amount of total protein applied in each lane was adjusted by the amount of β-actin. *p b 0.05.
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adjusting the loaded protein level by β-actin expression (p b 0.05, Fig. 3C). ECS treatment significantly enhanced GPR56 expression in the same region. The amount of GPR56 protein was 266% ±33% in the ECS-treated group (n =4) compared to 100% ±16% in the control group (n = 4), after adjusting the loaded protein level by β-actin expression (pb 0.05, Figs. 3A and C). The amount of nestin protein was 92%± 4% in the ECS-treated group (n= 4) compared to 100% ±6% in the control group (n =4); no statistical difference was detected (p N 0.05, Figs. 3D and F). The amount of nestin protein was 133%± 31% in the CVS-treated group (n= 6) compared to 100% ±22% in the control group (n =6); no statistical difference was detected (p N 0.05, Figs. 3D and F).
2.3. Effects of ECS on BDNF expression in the LV wall adjacent to the CPu The effects of ECS on the LV wall including the ependymal cell layer and the SVZ adjacent to the CPu of the rat brain were confirmed by western blot analysis that demonstrated a significant induction of the BDNF protein in this region. The amount of BDNF protein was 739% ±172% in the ECS-treated group (n= 4) compared to 100%± 27% in the control group (n= 4) after adjusting the loaded protein level by β-actin expression (p b 0.05, Figs. 3G and H).
2.4. Effects of CVS and ECS on GPR56 and nestin mRNA levels in the hippocampus It has been reported that GPR56 transcript expression is restricted to specific cells in the hippocampal SGZ, and RT-PCR is one of the limited methods to detect GPR56 expression in the hippocampus. RT-PCR analysis revealed that CVS treatment significantly reduced GPR56 transcript expression in the hippocampus. The amount of GPR56 transcript was 49%± 5% in the CVS-treated group (n= 6) compared to 100%± 10% in the control group (n= 6), after adjusting the reverse transcribed total RNA level by the amount of amplified β-actin (p b 0.05, Figs. 4C and D). ECS treatment significantly enhanced GPR56 transcript expression in the hippocampus. The amount of GPR56 transcript was 142%± 11% in the ECS-treated group (n =4) compared to 100% ±15% in the control group (n= 4), after adjusting the reverse transcribed total RNA amount by the amount of amplified βactin (pb 0.05, Figs. 4A and B). The amount of nestin transcript was 104% ±11% in the ECS-treated group (n= 4) compared to 100%± 17% in the control group (n= 4); no statistical difference was detected (p N 0.05, Figs. 4A and B). The amount of nestin transcript was 105% ± 6% in the CVS-treated group (n = 6) compared to 100% ±8% in the control group (n= 6); no statistical difference was detected (p N 0.05, Figs. 4C and D).
Fig. 4 – RT-PCR amplification of the hippocampal transcripts. [A and B] The 10-day electroconvulsive seizure (ECS) treatment induced significantly enhanced GPR56 transcript expression in the rat hippocampus without changing nestin transcript expression. The relative amount of total RNA used for RT-PCR was adjusted by the amount of amplified β-actin. In total, 30, 30 and 20 PCR cycles were applied for the amplification of nestin, GPR56 and β-actin, respectively. [C and D] Twelve-day chronic variable stress (CVS) treatment resulted in significantly reduced GPR56 transcript expression in the rat hippocampus without any changes in nestin transcript expression. The relative amount of total RNA used for RT-PCR was adjusted by the amount of amplified β-actin. In total, 25, 35 and 20 PCR cycles were applied for the amplification of nestin, GPR56 and β-actin, respectively. *pb b 0.05.
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The present study is the first to examine the immunohistochemical localization of the GPR56 protein around the LV. The results revealed that GPR56-positive cells express nestin but do not express Ki67, PSA-NCAM, NeuN or GFAP. The co-localization of GPR56 and nestin was different between the dorsal and ventral LV portions. In the dorsal portion of the ventricle, GPR56 immunoreactivity was limited to a line of the ependymal cell layer, whereas nestin immunoreactivity was widely distributed in the adjacent SVZ, particularly around the CPu. In the ventral portion of the ventricle, GPR56 protein expression completely coincided with nestin expression in the ependymal layer and SVZ. Restricted expression in only the ependymal cell layer around the CPu has been reported for a few proteins, including S100β (Kuo et al., 2006), notch1 (Johansson et al., 1999) and noggin (Lim et al., 2000). Nestin is a class IV intermediate filament protein and is downregulated during the process of differentiation from neural stem cells to neurons or glial cells (Yamaguchi et al., 2000; Fukuda et al., 2003). It has been reported that nestin is expressed prominently in ependymal cells around the ventricle and in putative precursor cells in the SVZ (Doetsch et al., 1997). Furthermore, in the present study, nestin immunoreactivity was observed in GPR56-positive ependymal cells and the Ki67-positive neuroblasts. Ernst and Christie (2005) reported that, in the SVZ, the total number of nestin-positive cells did not change despite the increased number of newly divided BrdU-incorporated cells after brain injury. In addition, in the SGZ, kainic acid induced seizures and brain ischemia robustly increase the cell division but do not increase the total number of nestin-positive cells (Yagita et al., 2002). These findings indicate that the total number of nestin-expressing cells does not correspond to mitotic activity in these areas, and this inference is in agreement with the present finding that the CVS and ECS treatments did not alter the amounts of nestin transcript or nestin protein in the hippocampus and lateral ventricular wall including the ependymal cell layer and the SVZ, respectively. Neither the total number of nestinpositive cells nor the amount of nestin expression appears to be a sensitive marker for estimating neural plasticity. Many studies reported that behavioral stress caused decreased neurogenesis in the hippocampal SGZ (Gould and Tanapat, 1999; Duman et al., 2001). Although there are abundant data for the hippocampus, only a few studies have examined the effects of stress on neurogenesis in the SVZ. Recent studies have indicated that the number of Ki67positive cells in the SVZ did not change after chronic restraint stress treatment (Kaneko et al., 2006a) and that the number of BrdU-labeled cells in the SVZ did not change after a chronic social stress paradigm (Czeh et al., 2007) and repeated electric tail shocks (Chen et al., 2006). Despite the previous discrepancy in the inhibition of neurogenesis in the SGZ and SVZ by stress exposure, the present study demonstrated a common observation of stress-induced decreases in GPR56 expression in both regions. In another study, bulbectomized rats, a classical model of depression whose aberrant behaviors improve with repeated antidepressant treatments (Breuer et al., 2007), showed reduced neurogeneis in the hippocampus as well
as around the LV (Keilhoff et al., 2006). It has been reported that the streptozotocin-induced diabetic model, which exhibits depressive features in various paradigms, showed decreased neurogenesis in both SGZ and SVZ (Saravia et al., 2004). Taken together, the effect of stress on the rate of neurogenesis in the SVZ still remains controversial. Further studies are required to elucidate the possible existence of a common mechanism that regulates the rate of neurogenesis in both SGZ and SVZ. Although we could not detect the translated GPR56 protein in the rat hippocampus, our RT-PCR examination revealed that the GPR56 transcript level changed significantly in accordance with the observations in the SVZ. We considered that GPR56 protein expression in the hippocampal SGZ is low compared to that in the ependymal cell layer and the SVZ around the LV. A previous study demonstrated limited GPR56 transcript expression in the SGZ of the hippocampus of the adult mouse (Piao et al., 2004). With regard to the SGZ and SVZ, the restricted neurogenic sites in the adult brain, it has been postulated that they share a similar microenvironment comprising radial glial cells, neural stem cells and ependymal cells in the SVZ or perivascular basal lamina in the SGZ (Alvarez-Buylla and Lim, 2004). It is known that BDNF transcript expression is upregulated by ECS and downregulated by behavioral stress loading (Nibuya et al., 1995). Furthermore, it has been shown that BDNF protein expression in the hippocampus is differentially regulated by the seizure (Nawa et al., 1995) and stress paradigm (Xu et al., 2002). Although a change in BDNF protein expression due to our stress paradigm could not be detected because of the low basal level in the SVZ homogenate, a robust increase in translation in the SVZ due to the 10-day ECS treatment was clearly demonstrated by western blotting. Intraventricular administration of BDNF has been reported to increase neurogenesis in the SVZ (Zigova et al., 1998). The present study raises a hypothesis needing further evidences in future studies that GPR56 expression in both SVZ and SGZ is commonly regulated by the same neurotrophin pathway involving BDNF secretion. The microenvironment of neurogenesis around the LV is comprised of ependymal cells, astrocytic B cells that transform to neural precursor C cells and neuroblastic A cells (Doetsch et al., 1997). It has been shown that, among these cells, only the ependymal cells are proliferatively silent in the adult brain (Doetsch et al., 1999; Spassky et al., 2005). Presently, there is growing evidence that the ependymal cell layer plays a major role in regulating cell fate and neurogenesis in the SVZ by differentially expressing various signaling molecules; these molecules include notch1 (Johansson et al., 1999), CD24 (Belvindrah et al., 2002), numb (Kuo et al., 2006), S-100β and noggin (Lim et al., 2000). Among these proteins, dramatic upregulation of CD24 has already been reported in the hippocampal SGZ after a seizure treatment (Elliott et al., 2003), and intraventricular infusion of S-100β has been reported to increase neurogenesis in the hippocampus (Kleindienst et al., 2005). Notch1 and noggin are considered to play a role in determining the fate of progenitor cells and regulating the rate of neurogenesis in the SVZ (Chambers et al., 2001; Lim et al., 2000). The notch1 protein is considered a cell surface receptor protein (Schroeter et al., 1998), and the present study raises the possibility that the GPR56 receptor protein is also involved in the regulatory process of neural plasticity. To exclude the possibility that the presently used antibody binds to other proteins in the immunohistochemical
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studies, its specificity must be tested using GPR56-knockout tissues in future studies. Taken together, the present study showed that the dynamically regulated expression of the GPR56 receptor protein in ependymal cells and hippocampus might be involved in the signaling of neural plasticity; this signaling might regulate neurogenic activity in the SVZ and SGZ in response to various physiological and environmental changes. The role of the GPR56 protein in both these adult brain regions in relation to stressrelated disorders, including depression and PTSD, should be elucidated by future studies.
Day 11: Cold stress was applied for 3 h at 5 °C, and crowding stress was applied for 18 h. Day 12: Forced cold swim stress was applied for 5 min, and vibratory stress was applied for 3 h. The abovementioned paradigm was used to examine the effect of preloaded chronic stress on a PTSD animal model (Wakizono et al., 2007) and was modified from previous methods that used a 10-day (Ortiz et al., 1996) or 14-day stress paradigm (Lu et al., 2006; Munhoz et al., 2006).
Male Wistar rats (Clea Inc, Tokyo, Japan) were used for all experiments. Three to four rats were housed per cage under controlled conditions (room temperature (RT), 21 °C; humidity, 55%–65%; and light cycle, 08:00 to 20:00 h) with food and water freely available. All animal treatments were in accordance with the Methods and Welfare Considerations in Behavioral Research with Animals (NIH: http://www.nimh.nih.gov/researchFunding/ animals.cmf), and the study was approved by the local Animal Investigation Committee of the National Defense Medical College. For immunostaining, 7- to 8-week-old rats were used (n =6). In both chronic variable stress (CVS) and electroconvulsive seizure (ECS) protocols, 5-week-old rats were used, and they were randomly assigned to control (n= 6), CVS (n =8), sham ECS (n =4), or ECS (n= 4) groups.
The CVS protocol
Rats were exposed to multiple stressors for 12 days as follows: Day 1: Cold (5 °C) and crowded stresses were applied (8 rats were housed in a cage with a dimension of 42 × 25 × 20 cm) for 3 h. Subsequently, the crowding stress was reapplied under the same conditions at room temperature except that the duration was increased to 18 h (overnight). Day 2: Forced cold swim stress was applied for 3.5 min at 12 °C in 400 mm deep water followed by water and food deprivation for 18 h. Day 3: Vibratory stress was applied to the cage using a seesaw-type rotor, which tilted the cage at a maximal angle of (±) 20° from the horizontal at a frequency of 60/min for 3 h, and the crowding stress was then applied for 18 h. Day 4: Cold stress was applied for 3 h at 5 °C followed by water and food deprivation for 18 h. Days 5–7: Forced cold swim stress was applied for 5 min, and crowding stress was applied for 66 h. Day 8: Vibratory stress was applied for 3 h followed by water and food deprivation for 18 h. Day 9: Cold stress was applied for 3 h at 5 °C, and vibratory stress was applied for 3 h. Day 10: Forced cold swim stress was applied for 5 min followed by water deprivation for 18 h (overnight).
The ECS protocol
A typical tonic–clonic seizure lasting for 7 s to 9 s was induced by using a pulse generator (Muromachi, Tokyo, Japan) via moistened bilateral earclip electrodes for 0.5 s (40 mA on the first 5 days and 50 mA on the following 5 days) once daily for 10 days. Shamtreated animals (sham ECS) were handled identically as the ECStreated animals but they received no electrical stimulation. The rats were killed at 3 h after the last treatment for analyses of proteins and transcripts.
Rats were decapitated after anesthetization with an intraperitoneal injection of 50 mg/kg sodium pentobarbital. The extracted brain was washed once in 1× PBS (137 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.2) and frozen in powdered dry ice. Coronal sections of 10 μm thickness containing the LV adjacent to the caudate putamen (CPu) were cut in a cryostat CM 1850 (Leica, Nussloch, Germany) and thawmounted onto adhesive MAS-coated slides (Matsunami, Osaka, Japan). Sections were fixed with 4% paraformaldehyde in 1× PBS for 5 min and blocked with 10% fetal bovine serum (FBS) in 1× PBS at RT for 60 min. Then, they were incubated overnight at 4 °C in 1× PBS with 2.5% FBS with one of the following primary antibodies: mouse monoclonal anti-nestin IgG (MAB353, Chemicon, Temecula, CA, USA; diluted at 1:400); mouse monoclonal anti-glial fibrillary acidic protein (GFAP) IgG (G3893, SigmaAldrich, St. Louis, MO, USA; diluted at 1:400); mouse monoclonal anti-neuronal-specific nuclear protein (NeuN) IgG (MAB377, Chemicon; diluted at 1:200); mouse monoclonal anti-polysialic acid-neural cell adhesion molecule (PSA-NCAM) IgG (MAB5324, Spring Biosciences Fremont, CA, USA; diluted at 1:1000); and rabbit anti-Ki67 polyclonal antibody (M3064, Spring Biosciences; diluted at 1:80000). After washing with 1× PBS (5 min × 5 times), the slides were incubated in 1× PBS for 60 min with a secondary antibody, anti-mouse or anti-rabbit IgG conjugated with Alexa Fluor 546 (Molecular Probes, Eugene, OR, USA; diluted at 1:200). The sections were then incubated with rabbit anti-GPR56 polyclonal IgG (LSA-1213, 15 μg/ml), a primary antibody purchased from MBL International (Woburn, MA, USA), in 1× PBS with 2.5% FBS for 90 min at RT. After washing with 1× PBS (5 min× 5 times), the slides were then incubated for 60 min at RT with anti-rabbit IgG conjugated with Alexa Fluor 488, a secondary antibody (Molecular Probes, diluted at 1:200). The immunologically stained slides were analyzed using a multifluorescence microscope BX51 (Olympus, Tokyo, Japan), and images were captured using a multi-channel camera DP71 (Olympus).
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4.5. Reverse transcribed-polymerase chain reaction (RT-PCR) The whole hippocampal regions were dissected and were immediately frozen on dry ice. After obtaining the total RNA by using the guanidium-isothiocyanate/cesium chloride ultracentrifugation method, the RNA was reverse transcribed with poly-T primers using a SuperScript First-Strand Synthesis System (Invitrogen, Groningen, Netherlands). The target cDNA was amplified using the following primers: β-actin sense primer, 5′-CCA GGG TGT GAT GGT GGG TA-3′ (corresponding to the 201–220 plus strand of NM031144); β-actin antisense primer, 5′-TAC GAC CAG AGG CAT ACA GG-3′ (523– 504 minus strand); GPR56 sense primer, 5′-GCC TCC AAC CTC CTC TGC TAC CGG-3′ (corresponding to the 635–658 plus strand of NM152242.1); GPR56 antisense primer, 5′-GCA GAT CCT CCA GCC CGG CTG TGG G-3′ (1037–1013 minus strand); nestin sense primer, 5′-GGG ACT GAG GCC TCT CTT CTT CCA GGG-3′ (corresponding to the 4951–4977 plus strand of AF538924); and nestin antisense primer, 5′-CCC CCT CCA AGG AAG CAG ACT CAG AC-3′ (5353–5328 minus strand). For amplification, 20 cycles for β-actin, 25 or 30 cycles for nestin and 30 or 35 cycles for GPR56 under thermal conditions of 94 °C for 30 s, 58 °C for 30 s and 72 °C for 1 min were applied. The amplified cDNAs were electrophoresed, and the calculated area × density of each band was quantified. All GPR56 data were normalized using the β-actin data.
Brains without hippocampal regions were washed with icecold 1× PBS and the LV wall, including the ependymal cell layer and the SVZ adjacent to the CPu, was dissected and lysed in a buffer containing 50 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100 and 10 μg/ml aprotinin. Following incubation on ice for 30 min, the lysed cells were centrifuged at 15,000×g for 20 min at 4 °C to precipitate debris. The supernatant was collected, and the protein content of these fractions was determined using a Protein Assay Reagent Kit (Pierce, Rockford, IL, USA). The samples were electrophoresed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred electrically to polyvinylidene difluoride membranes (Amersham, Arlington, IL, USA) by loading at 15 V for 90 min. After blocking the membrane with 4% skimmed milk for GPR56, BDNF and β-actin or with 5% bovine serum albumin for nestin for 1 h at room temperature, the membranes were reacted with specific antibodies overnight at 4 °C. The following antibodies were used: rabbit anti-GPR56 antibody (MBL; diluted at 1:1000), rabbit anti-BDNF antibody sc-546 (Santa Cruz Biotech, Santa Cruz, CA, USA; diluted at 1:400) and mouse anti-nestin antibody (Chemicon; diluted at 1:2000). The blots were washed and incubated for 1 h at RT with horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibodies NA9310 (Amersham Biosciences; diluted at 1:1000) or HRP-conjugated anti-rabbit IgG antibody #7074 (Cell Signaling Technology, Beverly, MA, USA; diluted at 1:1000). Signals were detected using a chemiluminescence ECL detection kit (Amersham). For immunoblotting of β-actin, the membranes were reprobed with A5441, a mouse anti β-actin antibody (Sigma-Aldrich; diluted at 1:5000), using standard methods.
Acknowledgment We wish to thank Ms. Sachiko Moroka for technical and secretarial assistance.
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