Sprouting of Nervous Fibers and Upregulation of C-X-C Chemokine Receptor Type 4 Expression in Hippocampal Formation of Rats with Enhanced Spatial Learning and Memory.код для вставкиСкачать
THE ANATOMICAL RECORD 295:121–126 (2012) Sprouting of Nervous Fibers and Upregulation of C-X-C Chemokine Receptor Type 4 Expression in Hippocampal Formation of Rats with Enhanced Spatial Learning and Memory BAOGUI SU,1 SANQIANG PAN,1* XIN HE,2 PENG LI,2 AND YINJI LIANG1 1 Department of Anatomy, Medical College of Jinan University, Guangzhou, People’s Republic of China 2 Department of Anatomy, Medical College of Beihua University, Changchun, People’s Republic of China ABSTRACT Hippocampal mossy ﬁber sprouting following training in the Morris water maze (MWM) is associated with spatial learning, memory and neural plasticity. The C-X-C chemokine receptor type 4 (CXCR4) is the main receptor for stromal cell-derived factor-1 (SDF-1), which is a chemokine that can regulate axonal elongation. This study aimed to investigate the relationship between the morphological plasticity of hippocampal formation and CXCR4 expression. A model of spatial learning and memory was established in rats by training using the MWM. Mossy ﬁber sprouting in the striatum oriens of the CA3 area of the hippocampus was found in trained rats by Neo-Timm’s method. As shown by immunohistochemistry, the CXCR4 immunopositive neurons were distributed in all layers and areas of hippocampal formation. There were no differences among groups regarding the distribution or shape of the immunopositive neurons. However, the immunoreactive staining intensity was increased in trained rats as compared with the control rats. Both CXCR4 gene transcription and translation were signiﬁcantly upregulated in the trained group as compared with the control group (P < 0.01). Morphological plasticity in the form of axonal sprouting in the hippocampal formation can be induced by enhanced spatial learning and memory activity, and CXCR4 mRNA and protein expression is upregulated, indicating a positive correlation between CXCR4 expression and axonal sprouting. Anat Rec, 295:121–126, C 2011 Wiley Periodicals, Inc. 2012. V Key words: spatial learning and memory; CXCR4; rat Neural plasticity in the mammalian central nervous system has recently received much attention (Cheung et al., 2005; Rabdoph, 2006). Many studies have shown that structural and/or functional reorganization of the nervous system can occur under pathological or physiological conditions (Zinck et al., 2006; Deller et al., 2007). The fact that tactile stimulation can induce functional reorganization of the primary somatosensory cortex in adult animals is of interest because the results indicate that rats raised in enriched environments have larger brains and increased cortical thickness (Jenkins et al., C 2011 WILEY PERIODICALS, INC. V 1990). Additionally, it has been shown that raising rats in enriched environments can result in larger neuronal *Correspondence to: Sanqiang Pan, Department of Anatomy, Medical College of Jinan University, Guangzhou, People’s Republic of China. E-mail: email@example.com Received 3 May 2011; Accepted 15 September 2011 DOI 10.1002/ar.21518 Published online 5 December 2011 in Wiley Online Library (wileyonlinelibrary.com). 122 SU ET AL. cell bodies and nuclei, synaptic contacts and dendritic spine density, and dendritic branching as well as a higher synapse to neuron ratio (Mohammed et al., 2002). These changes appear to be much greater and more rapid in rats that are exposed to enriched environments in early developmental periods. Until the 1960s, it was speculated that similar changes could occur in adult rats (Hannigan et al., 2007), and it was recently discovered that enriched environments result in enhanced neurogenesis in aged rat hippocampal formation (Laviola et al., 2008), which suggests that nervous system plasticity exists throughout animal life. We have previously reported mossy ﬁber sprouting (Su et al., 2000), synaptic plasticity (Su and Xu, 2003), increased synapsin (Su et al., 2000), and stromal cellderived factor-1 (SDF-1) expression (Li et al., 2008) in rats that have received compulsory training to enhance their memory and spatial learning. Because plasticity of the nervous system has been observed under multiple conditions, studies of its underlying mechanisms are a hot spot in neuroscience research. Axonal guidance factors were reported to play an important role in neural plasticity (Xu et al., 2003). Recently, it has been reported that SDF-1 and C-X-C chemokine receptor type 4 (CXCR4) are involved in neuronal migration and axonal pathﬁnding regulation (Stumm and Höllt, 2007). Consistent with these ﬁndings, SDF-1 expression is increased in the hippocampus of rats with enhanced spatial learning and memory (Berger et al., 2007). While it is recognized that CXCR4 is the main SDF-1 receptor, the CXCR4 response to morphological plasticity in the hippocampal formation remain unclear. Hence, in this report, we examine the hypothesis that morphological plasticity occurs in the hippocampal formation, and it is related to the CXCR4 expression in rats in a compulsory training model using the Morris water maze (MWM) to enhance spatial learning and memory. First, we established a MWM animal model for enhanced training of spatial learning and memory. Using this system, we examined mossy ﬁber sprouting using Neo-Timm’s staining and the shape and distribution of CXCR4 immunopositive cells with immunohistochemistry. Finally, we assessed CXCR4 mRNA and protein expression using reverse transcription-polymerase chain reaction (RTPCR) and Western blot, respectively. All results were analyzed to explore the relationship between the plasticity of hippocampal formation and CXCR4. MATERIALS AND METHODS Materials Male Sprague-Dawley rats were provided by the Guangdong medical experimental animal center. All animal experiments and procedures were approved by the animal welfare committee of Jinan Medical College. Rats were randomly assigned into the model, mock, or control groups. The model group rats (N ¼ 39) were trained for up to 21 days with a modiﬁed MWM protocol to enhance the spatial learning and memory capability of the animals. The mock group rats (N ¼ 13) were trained at the same water temperature and depth as the MWM model group, but they were allowed to swim randomly without any directional cues for 10 sec. The control group rats (N ¼ 13) received no compulsory training at all. The rats were anaesthetized with chloral hydrate and intracar- dially perfused with 4% paraformaldehyde. The brains were removed from the cranial cavity and separated into two halves at the middle longitudinal ﬁssure. The right halves of the brains (N ¼ 8) were sectioned at 50 lm with a vibrotome and then processed for use in NeoTimm’s staining. The left halves of the brains (N ¼ 8) were used for immunohistochemistry following postﬁxation with 4% paraformaldehyde. Every third section (10 lm) cut using a cryostat microtome was used for immunohistochemistry. The hippocampal homogenates (N ¼ 5) were prepared for Western blotting and RT-PCR. MWM Established Model The behavior of the rats from the MWM model group was analyzed with the digital behavior system. Prior to the training, the rats were placed in the maze and allowed to swim freely in the pool for 2 min. This compulsory training process occurred 12 times a day for six consecutive days. In each training session, the rats were randomly placed in a quadrant of the pool, and the time of escape from the water to the step (the escape latent period) was simultaneously recorded, and 60 sec was the upper limit. If the rat did not ﬁnd the step by 60 sec, the rat was assisted by guiding to reach the step, and then 60 sec was recorded as the escape latent period. The rats were allowed to rest on the step for 60 sec before repeating the training. The model of spatial learning and memory was established when the average run time was less than 20 sec, the average latent period was less 5 sec, and the run trace focused on just one quadrant. Neo-Timm’s Staining The sections taken from the brains and incubated with sodium sulﬁde were placed in Timm’s staining solution (100 mL) containing sodium sulﬁde, 50 mL arabic mucilage (50%), 22 mL phosphate buffer (7.65 g citric acid and 7.05 g sodium citric acid), and in 1.5 mL silver nitrate (17%). The staining was performed with shaking at 26 C for 80 min on a shaker. The sections were rinsed with distilled water, dehydrated, and mounted with neutral balsam. Immunohistochemistry The sections were incubated using an anti-CXCR4 antibody (1:200, Bo shi de company, Wuhan) following the protocol of the manufacturer. After the primary antibody incubation, the proteins were visualized using a reaction kit (Maxin company, Fuzhou). Nonspeciﬁc staining was determined in negative control sections in which phosphate buffered saline replaced the primary antibody. The shape and location of CXCR4 immunopositive cells were observed under light microscope. Western Blotting Proteins from the hippocampal tissues (N ¼ 5) were extracted by preparation of modiﬁed radioimmunoprecipitation buffer (50 mmol/L Tris-HCl, pH 7.5, 1% NP40, 0.5% sodium deoxychloric acid, 0.1% sodium dodecyl sulfate [SDS]) containing protease inhibitors. The Bradford method (Bio-Rad, USA) was used to assess the protein concentration. Totally, 50 lg of cerebral protein were separated by 10% SDS-PAGE, and the proteins were FIBER SPROUTING AND CXCR4 EXPRESSION 123 Fig. 1. Neo-Timm’s staining showing silver granular deposition of the hippocampal formation in the control group (A), the mock group (B), 7-day trained rats (C), 14-day trained rats (D), and 21-day trained rats (1E). The arrow indicates the sliver particles. DG, dentate gyrus; SO, stratum oriens; SP, stratum pyramidale; SL, stratum lucidum; PML, polymorphic layer. transferred onto a polyvinylidene ﬂuoride membrane. The membrane was ﬁrst blocked with 10% skim milk in Tris buffer saline Tween-20 buffer (25 mmol/L Tris-HCl, pH 7.4, 137 mmol/L KCl, 0.2% Tween-20) for 1 hr at room temperature followed by incubation with a primary antibody against CXCR4 (1:200, DAKO, Denmark). The immune complexes were detected by ECL (Cell Signaling Technologies, Danvers, MD, USA) and exposed to X-ray ﬁlm. The density of each band in the digitized images of the same gel was analyzed with an ultra-lum 8.0 gel analysis system. 1.2% agarose gradient gel electrophoresis along with molecular weight markers for reference. The resultant bands were visualized with UV light, and the relative intensity was quantiﬁed using a scanning densitometer. All values were normalized to the expression of the housekeeping gene. Reverse Transcription-Polymerase Chain Reaction RNA extraction was performed according to the protocol of the manufacturer (Perkin Elmer, USA). Totally, 1 lg of RNA was added to a tube containing 5 mM MgCl2, 1 mM of each dNTP, 50 mM KCl, 10 mM Tris-HCL, pH 8.3, 2.5 lM oligo dT, 20 units of RNAse inhibitor, and 50 units of moloney murine leukemia virus reverse transcriptase. The mixture was incubated at 45 C for 35 min, heated to 95 C for 5 min and placed on ice until used for PCR. The newly synthesized cDNA was then PCR ampliﬁed using primers speciﬁc for the targeted genes, along with a housekeeping gene, glucose-3-phosphate dehydrogenase (GPDH) or b-actin, which was used as a normalization control. Each reaction mixture contained 10 lL of cDNA in a ﬁnal concentration of 2 mM MgCl2, 10 mM Tris-HCL, pH 8.3, 50 mM KCl, 0.02 lM of each speciﬁc primer pair, and 2.5 units of Taq polymerase. The mixture was placed in a thermocycler for 30 cycles of 95 C for 30 sec, 60 C for 30 sec, and 74 C for 1 min. The PCR products were separated by 1– Statistical Analysis Data were presented as the means SEM. One-way analysis of variance was used to analyze the CXCR4 mRNA and protein expression with SPSS 10.0 software. A P < 0.05 by the Student’s t-test was considered statistically signiﬁcant. RESULTS Hippocampal Fiber Sprouting in the MWM Model Rats Hippocampal Timm-positive ﬁbers indicate mossy ﬁbers, which are demonstrated by brown–black silver deposition. Neo-Timm staining in sections from the control group revealed that the hippocampal formation demonstrated a silver deposition in the polymorphic layer of the dentate gyrus and stratum lucidum in the CA3 (Fig. 1A). The mock group results appeared no different from those of the control group (Fig. 1B). In the model group, newly formed silver grains were found at the stratum oriens of the CA3 region of hippocampal formation and in 21 out of 24 (87.5%) rats. The silver grains in the stratum oriens of CA3 region presented as a granular or lamellar shape in rats trained for 7 days (Fig. 1C), a lamellar or banded shape in rats trained for 14 days (Fig. 1D), and a point or granular shape in rats trained for 124 SU ET AL. Fig. 2. CXCR4 immunopositive neurons are distributed in the hippocampal formation in the control group. A: hippocampal formation; B: CA1; C: CA2; D: CA3; E: CA4; F: DG. ~ shows that the CXCR4 immunopositive product is localized in the cell membrane. The aster- isk indicates the round cells, the black arrows indicate the fusiform cells, and the white arrows point to the triangular cells. ML, molecular layer; GL, granule cell layer. The other abbreviations used in this ﬁgure are the same as in Fig. 1. 21 days (Fig. 1E). The silver grain staining in rats trained for 14 days was stronger than that in rats trained for 7 days; however, the staining was decreased in 21-day trained rats. expression at 14 days post-training was highest in the model group. These differences are statistically signiﬁcant (P < 0.01). Localization and Shape of CXCR4 Immunopositive Neurons In the control group, the CXCR4 immunopositive neurons, which were distributed mainly in the granular layer of the dentate gyrus and the pyramidal layer of the hippocampus, were round and ellipsoid or fusiform in shape. However, triangular neurons were distributed in the stratum oriens of the hippocampus and the polymorphic layer of the dentate gyrus. A few immunopositive neurons were found in the molecular layers of the hippocampus and dentate gyrus. The CXCR4 immunopositive reaction products were mainly localized in the cell membrane of round and ellipsoidal neurons (Fig. 2A–F). Interestingly, the CXCR4 immunopositive reaction product was found in the cytoplasm in the model group rats. CXCR4 immunopositive neurons showed a similar localization and shape among the different treatment groups; however, the degree of staining was stronger as compared with the control group (Fig. 3A–F). CXCR4 Gene Transcription and Protein Expression The differences in CXCR4 mRNA and protein expression between the three groups are shown in Fig. 4. The CXCR4 mRNA and protein expression in the model group was higher than that in the control and mock groups. Furthermore, the CXCR4 mRNA and protein DISCUSSION In this study, we found that enhanced learning and memory capability is associated with sprouting ﬁbers development in rats trained in the MWM. The mossy ﬁbers, which terminate in the stratum lucidum of the CA3 region, sprout into the stratum oriens of the CA3 region in 7-day trained rats. Whereas the sprouting ﬁbers from the dentate gyrus appeared greater after 14 days of training, they were reduced after 21 days of training. These results indicate that physiological activity related to enhanced spatial learning and memory can induce structural plasticity in the hippocampal formation, and that extent of sprouting is dynamic throughout the training period. SDF-1 has been deﬁned as a small, secreted leukocyte chemoattractant. It is also essential for the migration of cortical, cerebella and dentate gyrus neuronal precursors during central nervous system development (Zou et al., 1998; Lu et al., 2002; Borrell and Marin, 2006). In zebraﬁsh, the knockdown of SDF-1 or CXCR4 induced retinal axons that followed aberrant pathways in the retina (Li et al., 2005). These data have suggested that SDF-1/ CXCR4 signaling plays an important role in guiding retinal ganglion cell axons in the retina. We observed CXCR4 expression in pyramidal neurons of the hippocampus and granular neurons of the dentate gyrus. The CXCR4 immunostaining in the model group rats was stronger than that in the control group rats. Moreover, CXCR4 mRNA and protein expression were upregulated FIBER SPROUTING AND CXCR4 EXPRESSION 125 Fig. 3. CXCR4 immunopositive neurons are distributed in the hippocampal formation in the model group. A: Hippocampal formation; B: CA1; C: CA2; D: CA3; E: CA4; F: DG. ~ shows that the CXCR4 immunopositive product is localized in the cytoplasm. The asterisk indicates the round cells, the black arrows indicate the fusiform cells, and the white arrows point to the triangular cells. Fig. 4. The inﬂuence of spatial learning and memory enhanced activity on CXCR4 gene transcription and protein expression. RT-PCR and Western blot show that the CXCR4 mRNA and protein expression in the model group are higher as compared with the control and mock groups (A). Quantiﬁcation of CXCR4 gene transcription (B) and protein expression (C) among the groups. The data are expressed as the mean SEM values. *P < 0.01 versus the control group (b); *P < 0.01 versus 7 days and 21 days (c, N ¼ 5). in the model group where they peaked in the 14-day trained rats and decreased in the 21-day trained rats. The CXCR4 expression is consistent with hippocampal ﬁbers sprouting. Therefore, these data indicate that CXCR4 may be involved in the regulation of axonal pathﬁnding in the hippocampal formation. CXCR4 was considered the only SDF-1 receptor; however, CXCR7 has recently been identiﬁed as a SDF-1 126 SU ET AL. receptor as well (Burns et al., 2006). Supporting evidence has revealed that CXCR7 is involved in tumorigenesis (Maksym et al., 2009). CXCR7 knockout mice have demonstrated cardiac valve malformation or enlarged hearts (Leung et al., 2007; Gerrits et al., 2008), and no brain defects were observed in these animals. CXCR7 mRNA expression was considerably increased in the granular layer and in the CA3 pyramidal cell layer in the postnatal rat brain (Schönemeier et al., 2008). Thus, further studies are needed to explore whether CXCR7 is involved with mossy ﬁber sprouting during spatial learning and memory. Taken together, these results demonstrate that physiological activity from spatial learning and memory can induce hippocampal mossy ﬁber sprouting, and axonal sprouting is positively associated with CXCR4 expression. LITERATURE CITED Berger O, Li G, Han SM, Paredes M, Pleasure SJ. 2007. Expression of SDF-1 and CXCR4 during reorganization of the postnatal dentate gyrus. Dev Neurosci 29:48–45. Borrell V, Marin O. 2006. Meninges control tangential migration of hem-derived Cajal–Retzius cells via CXCL12/CXCR4 signaling. 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