Allowing animals to bite reverses the effects of immobilization stress on hippocampal neurotrophin expression

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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

Research Report

Allowing animals to bite reverses the effects of immobilization stress on hippocampal neurotrophin expression Taeki Lee a , Juri Saruta a , Kenichi Sasaguri a,⁎, Sadao Sato a , Keiichi Tsukinoki b a

Department of Craniofacial Growth and Development Dentistry, Kanagawa Dental College, Japan Department of Maxillofacial Diagnostic Science, Division of Pathology, Kanagawa Dental College, Japan

b

A R T I C LE I N FO

AB S T R A C T

Article history:

Acute immobilization stress alters the expression of neurotrophins, including brain-derived

Accepted 27 December 2007

neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), in rat hippocampus. We found that

Available online 15 December 2007

biting may be associated with reduction of systemic stress responses. The purpose of this study was to examine whether neurotrophin expression in rat hippocampus is influenced by biting.

Keywords:

Rats were exposed to immobilization stress for 2 h (stress group without biting) or biting for the

Brain-derived neurotropic factor

latter half of 2-hour immobilization stress (biting group). Adrenocorticotropic hormone (ACTH)

(BDNF)

and corticosterone levels were markedly elevated in the stress group, while the increases in

Neurotrophin-3 (NT-3)

ACHT and corticosterone were suppressed in the biting group. Decreased BDNF mRNA and

Hippocampus

increased NT-3 mRNA expression in hippocampus were detected on real-time polymerase

Acute stress

chain reaction (PCR) in the stress group. The decrease in BDNF mRNA under acute

Biting

immobilization stress was recovered by biting. In addition, the magnitude of increase in NT3 mRNA was decreased. No changes in expression of tyrosine receptor kinase B or C, the receptors for BDNF and NT-3, respectively, were observed in this model. These findings suggest that biting influences the alterations in neurotrophin levels induced by acute immobilization stress in rat hippocampus. © 2007 Elsevier B.V. All rights reserved.

1.

Introduction

The neurotrophins include NGF, BDNF, NT-3, NT-4/5, NT-6, and NT-7 (Lai et al., 1998; Lewin and Barde, 1996; Nilsson et al., 1998). Neurotrophins play key roles in neuronal survival, differentiation, connectivity, and plasticity (Lewin and Barde, 1996; Lu and Chow, 1999; McAllister et al., 1999; Schinder and Poo, 2000; Huang and Reichardt, 2001; Poo, 2001). BDNF is the most abundantly expressed neurotrophin in the mature central nervous

system (Hofer et al., 1990) and supports the survival of many types of neurons (Lindsay, 1996). BDNF also plays important roles in long-term potentiation (Dragunow et al., 1997; Figurov et al., 1996; Korte et al., 1996), dendritogenesis (McAllister et al., 1995), and activity-dependent neuroplasticity (Gall and Lauterborn, 1992; Rocomora et al., 1996). The effects of BDNF are mediated by TrkB (Lewin and Barde, 1996). NT-3 expression is particularly high during embryogenesis, and may influence the development of the hippocampus (Ernfors et al., 1990; Friedman

⁎ Corresponding author. 82 Inaoka-cho, Yokosuka 238-8580, Japan. Fax: +81 46 822 8858. E-mail address: [email protected] (S. Kenichi). Abbrevations: (NGF), nerve growth factor; (BDNF), brain-derived neurotrophic factor; (NT-3) neurotrophin-3; (NT-4/5), neurotrophin-4/5; (NT-6), neurotrophin-6; (NT-7), neurotrophin-7; (TrkB), tyrosine receptor kinase B; (TrkC), tyrosine receptor kinase C; (HPA), hypothalamic– pituitary–adrenal; (CRH), corticotropin releasing hormone; (PVN), paraventricular nucleus; (ACTH), adrenocorticotropic hormone; (GC), glucocorticoids; (NO), Nitric oxide;(nNOS), neuronal nitric oxide synthase 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.12.013

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et al., 1991). NT-3 mainly binds to TrkC (Lamballe et al., 1991). However, TrkB also binds weakly with NT-3. It is possible that BDNF and NT-3 have related effects in the hippocampus. Immobilization stress alters mRNA levels of neurotrophins such as BDNF and NT-3 in rat hippocampus (Ueyama et al., 1997). The major neuroendocrine response to stress is exerted via activation of the HPA axis, secretion of CRH by PVN of the hypothalamus, and subsequent release of ACTH from a population of anterior pituitary cells (Vreugdenhil et al., 2001). ACTH stimulates the adrenal glands to release GC, which are the primary means of regulatory control of the HPA axis (Bowman et al., 2001). GCs target the hippocampus (McEwen, 1999), a region of the brain that is intrinsically linked to learning and memory, and also interact with BDNF, a neurotrophic factor expressed at high levels in the hippocampus (Conner et al., 1997). On the other hand, it has been reported that biting modulates stress responses (Hori et al., 2004, 2005; Sasaguri et al., 2005). Expression of CRH is significantly increased in the PVN of the hypothalamus by immobilization stress, and this increase in CRH expression has been found to be suppressed by biting (Hori et al., 2004). NO modulates the activity of the endocrine system in behavioral responses to stress, and increase in nNOS mRNA expression under immobilization stress was observed, while biting of a wooden stick during immobilization decreased nNOS mRNA expression in the hypothalamus (Hori et al., 2005). Fos protein, expression of which is induced by acute immobilization stress, is generally used as a marker for neuronal activity in the PVN, while biting behavior during stress reduced the expression of Fos protein (Sasaguri et al., 2005). Biting can affect various stress-related molecules in the HPA axis. In this study, we therefore examined whether biting affects expression of neurotrophins such as BDNF and NT-3 and their receptors such as TrkB and TrkC within the rat hippocampus under conditions of acute immobilization stress.

2.

Results

2.1. Experiment 1: Stress hormone levels in control, stressed, and biting groups There were significant differences in ACTH level between the control and stress groups and between stressed and biting groups (p < 0.01 for all comparisons) (Fig. 1A). There were significant differences in corticosterone level between the control and stress groups and between the stressed and biting groups (p < 0.01 for all comparisons) (Fig. 1B).

2.2. Experiment 2: Quantitative analysis of BDNF, NT-3, TrkB, and TrkC mRNA There were significant differences in BDNF/β-actin ratio between the control and stress groups and between the stressed and biting groups (p < 0.05 for all comparisons) (Fig. 2A). There were significant differences in NT-3/β-actin ratio between the control and stress groups and between the stressed and biting groups (p < 0.01 for all comparisons) (Fig. 2B). There were no significant differences among groups in TrkB/β-actin ratio (Fig. 2C) or TrkC/β-actin ratio (Fig. 2D).

Fig. 1 – Stress hormone levels in 3 groups of rats (control, stressed, biting). (A) Plasma ACTH levels in 3 groups of rats (control, stressed, biting) Control group level was 91.25 ± 6.26 pg/ml, stress group 856.75 ± 27.54 pg/ml, and biting group 409.75 ± 9.13 pg/ml (n = 4, error bars = SEM). There were significant differences between the control and stressed groups and between the stressed and biting groups (**p < 0.01, ANOVA/Fisher's PLSD). (B) Corticosterone levels in 3 groups of rats (control, stressed, biting). Control group level was 99.25 ± 3.29 ng/ml, stress group 618.75 ± 8.15 ng/ml, and biting group 381.75 ± 17.21 ng/ml (n = 4, error bars = SEM). There were significant differences between the control and stressed groups and between the stressed and biting groups (**p < 0.01, ANOVA/Fisher's PLSD).

2.3. Experiment 3: Quantitative analysis of BDNF and NT-3 protein expression There were no significant differences among groups in BDNF levels (Fig. 3A). There were significant differences in NT-3 level between the control and stress groups and between the stressed and biting groups (p < 0.01 for all comparisons) (Fig. 3B).

3.

Discussion

Previous experimental research showed that biting has beneficial effects on stress-induced reactions in the PVN (Hori et al., 2004, 2005; Sasaguri et al., 2005). Although biting can affect various stress-related molecules such as c-Fos (Sasaguri et al., 2005), nNOS (Hori et al., 2005) and CRH (Hori et al., 2004), alteration of ACTH and corticosterone levels had not been examined in the biting condition. In this study, following acute immobilization stress, there were significant increases in ACTH and corticosterone levels relative to those in control rats. In addition, biting for the latter half of 2-hour stress significantly decreased both ACTH and corticosterone levels relative to those in stressed

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Fig. 2 – BDNF, NT-3, TrkB, and TrkC mRNA levels in rat hippocampus. (A) BDNF mRNA levels in 3 groups of rats (control, stressed, biting).Control group level was 0.547 ± 0.162, stress group 0.127 ± 0.028, and biting group 0.527 ± 0.120 (n = 4, error bars = SEM). There were significant differences between the control and stressed groups and between the stressed and biting groups (*p < 0.05, ANOVA/Fisher's PLSD). (B) NT-3 mRNA levels in 3 groups of rats (control, stressed, biting). Control group level was 0.044 ce:hsp sp="0.12"/>± 0.011, stress group 0.160 ± 0.037, and biting group 0.053 ± 0.009 (n = 4, error bars = SEM). There were significant differences between the control and stressed groups and between the stress and biting groups (**p < 0.01, ANOVA/ Fisher's PLSD). (C) TrkB mRNA levels in 3 groups of rats (control, stressed, biting). Control group level was 0.210 ± 0.012, stress group 0.210 ± 0.009, and biting group 0.206 ± 0.029 (n = 4, error bars = SEM). There were no significant differences among groups. (D) TrkC mRNA levels in 3 groups of rats (control, stressed, biting). Control group level was 0.337 ± 0.078, stress group 0.410 ± 0.094, and biting group 0.480 ± 0.077 (n = 4, error bars = SEM). There were no significant differences among groups.

rats. However, ACTH and corticosterone levels were unaffected by biting during the first half of 2-hour stress in the preliminary study (data not shown). 1-hour stress after biting seems to elevate the ACTH and corticosteron. Thus, biting for the latter half of 2-hour stress was an important situation to examine the effect of biting against the stress reaction. We demonstrated that biting influenced the ACTH and corticosterone levels in this stress model. Stress-induced activation of the HPA axis is characterized by enhanced expression of CRH in PVN and consequent increases in ACTH and adrenal glucocorticoid in plasma. Thus, the CRH expressed in the hypothalamus plays important roles in mediating behavioral responses to stressors. Restraining the body of an animal has been shown to activate and induce enhanced expression of CRH in the PVN of the rat hypothalamus (Givalois et al., 2004). On the other hand, biting during immobilization stress significantly suppressed stress-induced enhancement of CRH expression in the PVN (Hori et al., 2004). Previous studies and our findings suggest that stress reaction in the HPA axis may be reduced by biting. In the second part of our study, to examine alterations in BDNF and NT-3 in stress and biting conditions in the hippocampus, rats were exposed to acute immobilization stress for 2 h or allowed to bite during the latter half of 2-hour stress. Significant decrease in BDNF mRNA within the hippocampus was found in the stress group relative to control rats. The

biting condition significantly increased BDNF mRNA in rat hippocampus, relative to stressed rats. However, there was no significant decrease in BDNF protein within the hippocampus over time in the presence of this stress. This time course might be unaffected to BDNF protein level of hippocampus. On the other hand, there were significant increases in NT-3 mRNA and protein expression within the hippocampus in this stress condition, relative to those in control rats. The biting condition significantly decreased NT-3 mRNA and protein in rat hippocampus, relative to those in stressed rats. Interestingly, stress-induced increase in NT-3 expression may be associated with reduction of BDNF during immobilization stress (Hyman et al., 1994; Lindholm et al., 1994). In addition, intraventicular infusion of recombinant NT-3 caused a significant decrease in BDNF level of rat hippocampus (Ullal et al., 2007). Although it is not understood whether NT-3 modulated the BDNF in this study, it suggests that biting influences the hippocampal neurotrophins expression under the immobilization stress. Further, it needs to examine the relation between BDNF and NT-3 in the hippocampus. Acute (two-hour) and repeat immobilization stress have been shown to reduce BDNF mRNA level in the dentate, CA3, and CA1 areas of rat hippocampus, though acute stress did not affect NT-3 mRNA level while repeat stress increased NT-3 mRNA level in the CA2 and CA1 areas of rat hippocampus (Smith et al., 1995). When male rats were exposed to immobilization

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utilization and neural activity in hippocampus (McCloskey et al., 2001). Biting involves movement of masticatory muscles including the masseter muscle. In addition, biting can increase neuronal activities in various regions of the human brain including the insula, thalamus and cerebellum (Onozuka et. al., 2002). Exercise prevents the decrease in BDNF protein in the hippocampus following immobilization stress (Adlard et al., 2004). Thus, biting is likely to alter hippocampal neurotrophins of the male rats. We conclude that biting may prevent acute stress in the hippocampus.

4.

Experimental procedures

4.1.

Subjects and housing

.

Male Sprague–Dawley rats aged 7–9 weeks (Japan SLC, Shizuoka, Japan) and weighting 200–300 g were used in this study. Rats were group-housed (4 per cage) in a room maintained under controlled conditions of light (12:12 h light–dark cycle) and temperature (22 ± 3 °C). Animals had free access to food pellets and tap water. Fig. 3 – BDNF and NT-3 protein levels in rat hippocampus. (A) BDNF protein levels in 3 groups of rats (control, stressed, biting). Control group level was 16.083 ± 2.887, stress group 10.231 ± 1.427, and biting group 15.612 ± 0.388 (n = 4, error bars = SEM). There were no significant differences among groups. (B) NT-3 protein levels in 3 groups of rats (control, stressed, biting). Control group level was 106.498 ± 8.620, stress group 175.899 ± 21.209, and biting group 102.594 ± 4.708 (n = 4, error bars = SEM). There were significant differences between the control and stressed groups and between the stressed and biting groups (**p < 0.01, ANOVA/Fisher's PLSD).

stress for 8 h, significant decreased in BDNF mRNA was observed in the brain, especially in the CA3 and CA1 areas of the rat hippocampus, and NT-3 mRNA level was also decreased in CA2 (Ueyama et al., 1997). These findings were obtained using the in situ hybridization technique. Adlard et al. (2004) found that BDNF protein levels were not significantly decreased under acute immobilization stress for 2 h in mouse hippocampus as determined by ELISA. However, BDNF protein levels were significantly decreased at both 5 h and 10 h after cessation of stress. Acute and repeat immobilization stress clearly decreased BDNF level within the hippocampus, although the pattern of expression of NT-3 in these conditions is still unclear. In contrast, in neuropathies such as depression (Karege et al., 2005), schizophrenia (Tan et al., 2005) and Alzheimer's disease (Michalski and Fahnestock, 2003), decreased BDNF expression has been confirmed in the hippocampus. Decreased BDNF content in the hippocampus can play an important role in the pathogenesis of neural disease. Recent studies have shown increases in hippocampal BDNF protein after exercise in female rats and male mice but not change in BDNF level in female mice (Cesar et al., 2007). Effect of exercise includes increases in cerebral blood flow, glucose

4.2.

Immobilization stress and biting procedure

All experiments were performed with four rats per group. Control rats were not exposed to immobilization. Acutely restrained rats were immobilized to produce acute stress according to a well-established protocol (Hori et al., 2004, 2005). They were fixed on a wooden board (18 × 25 cm) in the supine position by a leather belt, after which each of their legs were fixed at an angle of 45 degrees to the body midline with adhesive tape. Immobilization stress was continued for 2 h (Smith et al., 1995). In a separate group, four rats exposed to 2-hour immobilization stress were allowed to bite a wooden stick (diameter, 0.5 cm) during the latter half of the immobilization period. The wooden stick was manipulated toward the rat's mouth, allowing the rat to bite it without any head or body movements. Rats of both the biting and stressed groups were killed immediately after immobilization stress. All experiments were performed with a total of 12 rats. The experimental protocol of this study was reviewed and approved by the Committee on Ethics on Animal Experiments of Kanagawa Dental College and performed under the Guidelines for Animal Experimentation of Kanagawa Dental College.

4.3.

Statistical analysis

Statistical analyses were carried out using the SPSS (Version 13.0) statistics program. All values are the mean± SEM. One-way analysis of variance (ANOVA) was performed following Fisher's PLSD test with findings of p < 0.05 considered significant.

4.4. Experiment 1: Preparation of plasma and measurement of stress hormones Plasma samples were obtained from the same rats as used for tissue preparation. Plasma samples were obtained from each rat's heart under anesthesia and collected in Venoject® tubes

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containing EDTA (TERUMO, Tokyo, Japan). The tubes were immediately placed on ice and then centrifuged (2000 r.p.m., 15 min, 4 °C). Adrenocorticotropic hormone (ACTH) was assayed using a radioimmunoassay (RIA) kit (ACTH IRMA MITSUBISHI, Mitsubishi Kagaku Yatoron, Tokyo, Japan) following the manufacturer's instructions. ACTH levels were reported in pg/ml. In addition, the COAT-A-COUNT Rat Corticosterone kit (Siemens Medical Solutions Diagnostics, New York, USA) was used for measurement of rat corticosterone (ng/ml).

4.5. Experiment 2: Tissue preparation, RNA Extraction, and cDNA synthesis, and real-time PCR 4.5.1.

Preparation of hippocampus

Animals were decapitated immediately after stress, the brain was removed, and the hippocampus dissected out. The right hippocampus was examined for expression of BDNF mRNA level by real-time PCR, while the left hippocampus was examined for expression of BDNF protein level by ELISA. These samples were stored at −80 °C before use.

4.5.2.

RNA extraction and cDNA synthesis

Total RNA isolation from the hippocampus was performed using ISOGEN reagent (Nippon Gene, Toyama, Japan) in accordance with the manufacturer's instructions. The RNA product was resuspended in 20 μl diethyl pyrocarbouate (DEPC)treated water. The quality of RNA was judged from the pattern of ribosomal RNA after electrophoresis of RNA through 1.5% agarose gel containing ethidium bromide (EB) and visualization by UV illumination. RNA concentrations were determined by absorbance readings at 260 nm with a SmartSpec Plus spectrophotometer (BIO-RAD, Tokyo, Japan). RNA was stored at −80 °C until use. Total RNA was reverse-transcribed at 50 °C for 30 min, 99 °C for 5 min, and 5 °C for 5 min using a single-strand cDNA synthesis kit (Roche Diagnostics Ltd., Lewes, UK) according to the instructions of the manufacturer of the reagent. Following the reverse transcription reaction, the cDNA products were stored at −20 °C until use.

4.5.3.

Real-time PCR analysis

Real-time PCR was performed using a LightCycler (Roche) according to the manufacturer's instructions. Reactions were performed in a 20 μl volume (BDNF: 0.3 μlM of each primer and 4 mM MgCl2; NT-3, TrkC: 0.5 μM of each primer and 3 mM MgCl2; TrkB: 0.15 μM of each primer and 4 mM MgCl2). Reactions with Taq DNA polymerase, nucleotides, and buffer for TrkC, TrkB, and BDNF were performed with LightCycler-DNA Master SYBR Green I mix, while those for NT-3 were performed with LightCyclerDNA Master HybProbe mix (Roche). Oligonucleotide primers were designed to amplify rat BDNF and were specific for the coding region of exon 5. The BDNF-specific primers were 5′-CAGGGGCATAGACAAAAG-3′(forward), 5′-CTTCCCCTTTTAATGGTC-3′ (reverse) (BDNF PCR production: 167 bp)(Tsukinoki et al., 2006, 2007); those for the TrkC-specific primer were 5′-CTTCCGCATGAACATCAGT-3′(forward), 5′-ACATTCACCAG(G,C)GTCAAGTT3′(reverse) (TrkC PCR production: 222 bp); those for the TrkBspecific primer were 5′-CACACACAGGGCTCCTTA-3′(forward), 5′-AGTGGTGGTCTGAGGTTGG-3′(reverse) (TrkB PCR production: 169 bp); and those for the NT-3-specific primer were 5′TGTGGGTAGCCGACAAGTC-3′(forward), 5′-GAGTTCCAGT-

47

GTTTGTCATC-3′(reverse) (NT-3 PCR production: 175 bp) as designed and synthesized by Nippon Gene Laboratory. Real-time PCR for amplification of the rat β-actin housekeeping gene was performed using a LightCycler Primer/Probe set, 5′-CCTGTATGCCTCTGGTCGTA-3′(forward), 5′-CCATCTCTTGCTCGAAGTCT3′(reverse) (β-actin PCR production: 260 bp), following the manufacturer's instructions (Nihon Gene Research Labs Inc., Sendai, Japan). Denaturation was performed at 95 °C for 10 min, after which Segment 1 (95 °C for 10 s), Segment 2 (60 °C for 10 s), and Segment 3 (72 °C for 10 s) were repeated for 40 cycles. We performed melting analysis and agarose gel electrophoresis to confirm the specificity of the PCR products obtained using each primer pair. Gene expression was expressed in terms of the ratio of copy number of BDNF, NT-3, TrkC, and TrkB to β-actin for each sample (mean±SEM).

4.6.

Experiment 3: Protein extraction and ELISA analysis

4.6.1.

Protein extraction

Whole hippocampal samples were homogenized in ice-cold lysis buffer, containing 137 mM NaCl, 20 mM Tris–HCl (pH 8.0), 1% NP40, 10% glycerol, 1 mM PMSF 10 μg/ml aprotinin, 1 μg/ml leupeptin, and 0.5 mM sodium vanadate. The tissue homogenate solutions were centrifuged at 14000 ×g for 5 min at 4 °C. The supernatants were collected and used for quantification of total protein and neurotrophin levels. Total protein concentrations were determined by the Bradford method using absorbance readings at 260 nm with a SmartSpec Plus spectrophotometer (BIO-RAD, Tokyo, Japan).

4.6.2.

Enzyme immunoassay for BDNF and NT-3

BDNF and NT-3 levels were assessed in the left hippocampus using an ELISA kit (Promega, Co., Madison, USA). Briefly, standard 96-well flat-bottom NUNC-immuno maxisorp ELISA plates were incubated with the corresponding captured antibody, which binds the neurotrophin of interest, overnight at 4 °C. The plates were blocked by incubation for 1 h at room temperature (RT) with a 1× block and sample buffer. Serial dilutions of known amounts of BDNF ranging from 0 to 500 pg/ ml were performed in duplicate for standard curve determination. Serial dilutions of known amounts of NT-3 ranging from 0 to 300 pg/ml were also performed in duplicate for standard curve determination. Wells containing the standard curves and supernatants of brain tissue homogenates were incubated at RT for 6 or 2 h, as specified by the protocol. They were then incubated with second specific antibody overnight at 4 °C or for 2 h at RT, as specified by the protocol. A species-specific antibody conjugated to horseradish peroxidase was used for tertiary reaction for 2.5 or 1 h at RT following this incubation step. TMB One Solution was used to develop color in the wells. This reaction was terminated with 1 N hydrochloric acid at a specific time (10–15 min) at RT, and absorbance was then recorded at 450 nm in a plate reader within 30 min of stopping the reaction. The neurotrophin values were determined by comparison with the regression line for each proposed neurotrophin standard. Using these kits, BDNF can be quantified in the range of 7.8–500 pg/ml and NT-3 in the range of 4.7– 300 pg/ml. For each assay kit, cross-reactivity with other neurotrophic proteins is < 2–3%.

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Acknowledgments This work was performed at The Research Institute on Occlusive Medicine of Kanagawa Dental College and supported by a Grantin-aid for Open Research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology.

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