close

Вход

Забыли?

вход по аккаунту

?

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: tpsq@jnu.edu.cn
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.
Nat Neurosci 9:1284–1293.
Burns JM, Summers BC, Wang Y, Melikian A, Berahovich R, Miao Z,
Penfold ME, Sunshine MJ, Littman DR, Kuo CJ, Wei K, McMaster
BE, Wright K, Howard MC, Schall TJ. 2006. A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion,
and tumor development. J Exp Med 203:2201–2213.
Cheung SW, Nagarajan SS, Schreiner CE, Bedenbaugh PH, Wong
A. 2005. Plasticity in primary auditory cortex of monkeys with
vocal production. J Neurosci 25:2490–2503.
Deller T, Del Turco D, Rappert A, Bechmann I. 2007. Structural reorganization of the dentate gyrus following entorhinal denervation: species
differences between rat and mouse. Prog Brain Res 163:501–528.
Gerrits H, van Ingen Schenau DS, Bakker NE, van Disseldorp AJ,
Strik A, Hermens LS, Koenen TB, Krajnc-Franken MA, Gossen
JA. 2008. Early postnatal lethality and cardiovascular defects in
CXCR7-deficient mice. Genesis 46:235–245.
Hannigan JH, O’leary-Moore SK, Berman RF. 2007. Postnatal environmental or experiential amelioration of neurobehavioral effects
of perinatal alcohol exposure in rats. Neurosci Biobehav Rev
31:202–211.
Jenkins WM, Merzenich MM, Ochs MT, Allard T, Guı́c-Robles E.
1990. Functional reorganization of primary somatosensory cortex
in adult owl monkeys after behaviorally controlled tactile stimulation. J Neurophysiol 63:82–104.
Laviola G, Hannan AJ, Macrı̀ S, Solinas M, Jaber M. 2008. Effects
of enriched environment on animal models of neurodegenerative diseases and psychiatric disorders. Neurobiol Dis 31:159–
168.
Leung H, Groom J, Batten M, Harvey RP, Martı́nez-A C, Mackay
CR, Mackay F. 2007. Disrupted cardiac development but normal
hematopoiesis in mice deficient in the second CXCL12/SDF-1 receptor, CXCR7. Proc Natl Acad Sci USA 104:14759–14764.
Li Q, Shirabe K, Thisse C, Thisse B, Okamoto H, Masai I, Kuwada
JY. 2005. Chemokine signaling guides axons within the retina in
zebrafish. J Neurosci 25:1711–1717.
Li Z, He X, Su BG, Wen QJ. 2008. Effect of spatial learning and
memory activity on morphology of SDF-1 positive cell in frontal
lobe cortex in rats. Anatomy Research Sinica 30:432–434.
Lu M, Grove EA, Miller RJ. 2002. Abnormal development of the
hippocampal dentate gyrus in mice lacking the CXCR4 chemokine
receptor. Proc Natl Acad Sci USA 99:7090–7095.
Maksym RB, Tarnowski M, Grymula K, Tarnowska J, Wysoczynski
M, Liu R, Czerny B, Ratajczak J, Kucia M, Ratajczak MZ. 2009.
The role of stromal-derived factor-1--CXCR7 axis in development
and cancer. Eur J Pharmacol 625:31–40.
Mohammed AH, Zhu SW, Darmopil S, Hjerling-Leffler J, Ernfors P,
Winblad B, et al. 2002. Environmental enrichment and the brain.
Prog Brain Res 138:109–133.
Rabdoph J. 2006. Plasticity. Neuron 3:420–427.
Schönemeier B, Kolodziej A, Schulz S, Jacobs S, Hoellt V, Stumm R.
2008. Regional and cellular localization of the CXCl12/SDF-1 chemokine receptor CXCR7 in the developing and adult rat brain. J
Comp Neurol 510:207–220.
Stumm R, Höllt V. 2007. CXC chemokine receptor 4 regulates neuronal migration and axonal pathfinding in the developing nervous
system: implications of neuronal regerneration in the adult brain.
J Mol Endocrinol 38:377–382.
Su BG, Pan SQ, Hang H, Yang XM. 2000. Change of synapsins in
hippocampal formation during spatial learning and memory in
rats. Chinese J Pathophysiol 16:421–423.
Su BG, Pan SQ, Hang H, Yang XM. 2000. Study on tracing the
sprouting fiber of the orients layer of CA3 subregion in hippocampus after spatial learning and memory in rats. Chinese J Anatomy 23:516–519.
Su BG, Xu LX. 2003. Morphological observation of synaptic plasticity of hippocampal formation induced by activity of spatial learning and memory. Chinese J Anatomy 26:35–39.
Xu B, Li S, Brown A, Gerlai R, Fahnestock M, Racine RJ. 2003.
EphA/ephrin-A interactions regulate epileptogenesis and activitydependent axonal sprouting in adult rats. Mol Cell Neurosci
24:984–999.
Zinck ND, Downie JW. 2006. Plasticity in the injury spinal cord:
Can we use it to advantage to reestablish effective bladder voiding and continence? Pro Brain Res 152:147–162.
Zou YR, Kottmann AH, Kuroda M, Taniuchi I, Littman DR. 1998.
Function of the chemokine receptor CXCR4 in haematopoiesis
and in cerebellar development. Nature 393:595–599.
Документ
Категория
Без категории
Просмотров
12
Размер файла
456 Кб
Теги
expressions, fiber, memory, formation, typed, learning, spatial, chemokine, nervous, hippocampus, enhance, upregulation, receptov, rats, sprouting
1/--страниц
Пожаловаться на содержимое документа