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1 Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Center Utrecht, Universtiteitsweg 100, 3584 CG Utrecht, The Netherlands
2 Altrecht – Rintveld Eating Disorders, Oude Arnehemseweg 260, 3705 BK, Zeist, The Netherlands
(Requests for offprints should be addressed to R A H Adan; Email: r.a.h.adan{at}med.uu.nl)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The activity-based anorexia (ABA) model is considered an animal model of AN. In this model, food access restricted to one hour per day in combination with voluntary access to running wheels is reported to result in hyperactivity, hypophagia and severe body weight loss (Hall & Hanford 1954, Routtenberg & Kuznesof 1967). In addition to hyperactivity, rats show an altered activity pattern and develop extensive activity prior to food access, which is referred to as food-anticipatory activity (FAA) (Mistlberger 1994). Furthermore, it was shown before that ABA rats are hypothermic (Hillebrand et al. 2005a) and have an increased activation of the hypothalamus-pituitary-adrenal (HPA) axis (Burden et al. 1993, Kas et al. 2003). In an alike semi-starvation-induced hyperactivity (SIH) model (which uses 60% of baseline food intake/day instead of 1 h food access/day), 5-HT turnover was increased in the medial basal hypothalamus (Broocks et al. 1991).
The melanocortin (MC) system plays an important role in energy homeostasis (Cone 1999, Schwartz et al. 2000). Expression levels of pro-opiomelanocortin (POMC) were decreased in the arcuate nucleus (ARC) of the hypothalamus of rats that were exposed to the ABA paradigm for one week (Kas et al. 2003). Recently, we observed that stimulation of the MC system by chronic treatment with the endogenous agonist 
-melanocyte stimulating hormone (-MSH, a neuropeptide derived from POMC), decreased food intake and increased FAA, body weight loss and activation of the HPA axis in ABA rats (Hillebrand et al. 2005b). Previously, we also showed that AgRP(83–132) treatment prevented self-starvation in ABA rats, by influencing food intake, locomotor activity and body temperature (Kas et al. 2003).
D-fenfluramine (d-FEN) stimulates 5-HT release and blocks its reuptake (Rowland & Carlton 1986, Gibson et al. 1993, McCann et al. 1997). The 5-HT1B and 5-HT2C receptor are the main candidate receptors mediating the inhibitory control of 5-HT on food intake. Both these receptors also seem to be involved in d-FEN-induced hypophagia (Dourish 1995, Tecott et al. 1995, Simansky 1996, Clifton et al. 2000, Vickers et al. 2003). 5-HT1B as well as 5-HT2C deficient mice showed an attenuated response to d-FEN-induced hypophagia, although they still had d-FEN-induced locomotor deficits at high doses (Lucas et al. 1998, Vickers et al. 1999, Lee et al. 2004). Hypophagic actions of d-FEN were reduced by co-treatment with a 5-HT1B or 5-HT2C receptor antagonist (Vickers et al. 2001, Simansky & Nicklous 2002). Yet other studies showed that mainly the 5-HT2C receptor plays an important role in d-FEN-induced hypophagia (Vickers et al. 2001, Clifton et al. 2003). It was recently demonstrated that 5-HT2C receptors are located on POMC neurons in the ARC. Activation of POMC neurons by d-FEN resulted in an increased firing rate of these neurons and subsequently in an anorexic response (Heisler et al. 2002).
Given that interference with the MC system influences the development and propagation of ABA (Kas et al. 2003, Hillebrand et al. 2005b) and since d-FEN acts upstream of the MC system (Heisler et al. 2002), we hypothesized that d-FEN treatment enhances ABA by reducing food intake and increasing RWA, specifically FAA. Although d-FEN-induced hypophagia in ABA rats was reported before (Rieg et al. 1994), effects of d-FEN treatment on running wheel activity (RWA) have not been observed. In the present study we investigated the effect of d-FEN treatment on development and propagation of ABA further, by analyzing several characteristics of ABA such as ingestive behaviour (food and water intake), RWA (including FAA), body temperature and activation of the HPA axis as well as hypothalamic POMC gene expression.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Female outbred Wistar WU rats (n=44, Harlan, Horst, The Netherlands) were individually housed in a temperature and humidity controlled room (21 ± 2 °C) under a 12:12 h dark:light cycle (ZT12=lights off). The ethical committee on use and care of animals of Utrecht University approved all described procedures.
Drugs
For in vivo administration, d-FEN (Servier, Paris, France) was dissolved in sterile isotonic saline and was chronically (continuous during 1 week) infused subcutaneously (sc) at a dose of 10 mg/kg/day using osmotic minipumps (0.5 µl/h, Alzet, model 1007D, DURECT Corporation, Cupertino, CA, USA). No adverse reactions to chronic administration of this dose of dFEN were reported before (Reig et al. 1994).
Surgical procedures
One week after arrival, rats received transmitters (TA10TA-F40 Data Sciences International, St Paul, MN, USA) in the abdominal cavity under fentanyl/fluanisone (Hypnorm®, Janssen Pharmaceutica, Beerse, Belgium, 0.1 ml/100 g intramuscular) and midazolam (Dormicum®, Hoffman-LaRoche, Mijdrecht, The Netherlands, 0.05 ml/100 g intraperitoneal) anaesthesia (ABA experiment only). After surgery, rats were treated with buprenorphin (Temgesic®, Schering-Plough, Maarssen, The Netherlands, 0.05 ml/100 g sc) and saline (1 ml sc).
For chronic vehicle or d-FEN infusions, all rats were anaesthetized by fentanyl/fluanisone as described above and the right flank was shaved. An osmotic minipump containing vehicle or d-FEN (10 mg/kg/day) was sc. placed into the flank region of the rat after overnight incubation at 37 °C.
Experimental set-up
d-Fenfluramine treatment in ad libitum fed rats
In this experiment, the effects of chronic d-FEN treatment in ad libitum fed sedentary rats were investigated. Two weeks after arrival (day –1), 14 rats were divided into two groups (vehicle and d-FEN), matched for body weight (average body weight day –1: 238.2 ± 2.5 g). Osmotic minipumps were implanted as described before. After surgery (day 0, ZT 12), food and water were ad libitum available. Each day, body weight, food intake and water intake were measured at the end of the light phase (ZT11). Rats were decapitated after 1 week (day 6, ZT11). Trunk blood was collected into lithium-heparin containing tubes (Sarstedt, Nümbrecht, Germany) after adding 83 µmol EDTA and 1 mg aprotonin. Plasma was separated and frozen at –20 °C. Brains were rapidly removed, quickly frozen in cold (–35 °C) isopentane and stored at –80 °C. Adrenal glands were isolated and weighed.
d-Fenfluramine treatment in activity-based anorexia rats
In this experiment, the effects of chronic d-FEN treatment on development and propagation of ABA were investigated. After 2 weeks of recovery from the implantation of transmitters, 16 rats were placed into cages with running wheels for a training period of 10 days (day –10 to day –1) with ad libitum food and water access. RWA was continuously registered using a Cage Registration Program (Department of Biomedical Engineering, UMC Utrecht, The Netherlands). On day –1, rats were divided into two groups (vehicle and d-FEN), matched for body weight (average body weight day –1: 227.7 ± 2.7 g) and baseline RWA (average day-4 : day-1: 4024.5 ± 510.9 revolutions/day). Transmitters were switched on (by magnetic field induction) to allow continuous assessment of body temperature and locomotor activity (LMA). Osmotic minipumps were implanted as described before. After surgery (day 0, ZT12), food was removed, while water remained ad libitum available. Food was available during the first hour of the dark phase (ZT12 to ZT13) (day 1–6). Each day, body weight, food intake and water intake were measured. Rats were decapitated after 1 week (day 6, ZT11) as described above.
In situ hybridization
Cryosections (coronal, 20 µm) of the hypothalamus of all rats were sliced using a cryostat (Leica, Rijswijk, The Netherlands) and thaw-mounted onto RNAse free Superfrost slides (Menzel, Brannschweig, Germany). The slides were stored at –80 °C until processed for in situ hybridization. Sections were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 10 min, washed in PBS, pre-treated with 0.25% acetic anhydride in 0.1 M triethanolamine, washed again in PBS and dehydrated in graded ethanol followed by 100% chloroform and 100% ethanol. 33P-labelled antisense RNA probes were made using a 350 bp rat POMC cDNA fragment and a 205 bp arginine-vasopressin (AVP) cDNA fragment (exon C) (Kas et al. 2003). The sections were hybridized overnight at 72 °C with 1 x 106 c.p.m. probe in buffer containing 50% deionized formamide, 2 x standard saline citrate (SSC), 10% dextrane sulphate, 1 x Denhardt’s solution, 5 mM EDTA and 10 mM phosphate buffer, after 5 min heating at 80 °C. After hybridization, the sections were washed in 5 x SSC (short, 72 °C) and 0.2 x SSC (2 h, 72 °C) and dehydrated in graded ethanol with 3 M ammoniumacetate. Sections were exposed to X-ray films (Kodak Bio-Max MR, Amersham) for 5 days. The films were developed and expression of POMC (in the ARC) and AVP (in the paraventricular nucleus (PVN) and supraoptic nucleus (SON)) was semi-quantitatively analyzed using a calibration curve and the Microcomputer Imaging Device (MCID) (Imaging Research Inc., St Catharines, Ontario, Canada).
Radioimmunoassay
Plasma levels of corticosterone, adrenocorticotropic hormone (ACTH) and leptin were analyzed by radioimmunoassay (RIA). A commercially available rat corticosterone RIA kit (ICN Biochemicals, Costa Mesa, CA, USA) and rat leptin RIA kit (Linco Research, St Charles, MO, USA, detection limit 0.5 ng/ml) were used. Assays were performed according to the manufacturer’s protocol. Plasma ACTH was measured using a specific rabbit antiserum directed to the midportion of ACTH, which was kindly provided by Dr G B Makara (Budapest, Hungary). Synthetic human ACTH(1–39) (Peninsula Laboratories, Belmont, CA, USA) was labelled with 125I and used as a tracer (Nijsen et al. 2000).
Plasma osmolality
Plasma osmolality was measured by U-Diagnostics (Utrecht, The Netherlands) using a Fiske® 2400 multi-sample osmometer according to the manufacturer’s protocol (Indumed, Dendermonde, Belgium).
Data analysis
All data are presented as mean±S.E. Data were analyzed using SPSS 11.5 (SPSS Inc., Amsterdam, The Netherlands) for Windows and were controlled for normality and homogeneity. For all measurements, baseline levels were not significantly different between groups. Basal body temperature was analyzed as average body temperature (of telemetric recordings) during 30 min of inactivity in the early light phase (ZT0-ZT3). Relative body weight, food intake, water intake, RWA (ABA only), LMA (ABA only) and basal body temperature (ABA only) were analyzed by general linear model (GLM) repeated measures using Huynh-Feldt correction for Mauchlys sphericity effects and followed by t-tests. Cumulative RWA and LMA (ABA only), cumulative water and food intake, HPA axis activation, plasma osmolality and ISH data were analyzed by t-tests. Differences were considered significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Ad libitum fed sedentary rats were treated with 10 mg/kg/day d-FEN by osmotic minipumps for one week. This treatment significantly influenced food intake (day F(6,72)=85.95; P < 0.001, day x treatment F(6,72)= 16.06; P < 0.001). d-FEN-treated rats had a reduced cumulative (day 0–6) food intake (t(12)=7.05; P < 0.001) as compared with vehicle-treated rats (Figure 1A
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). Body weight loss was also evident from reduced plasma leptin levels following d-FEN treatment (t(11)=2.97; P=0.01) (Table 1).
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). However, plasma osmolality was not changed following 1 week of d-FEN treatment (t(12)=–1.77; not significant, n.s.) and neither were AVP gene expression levels in the PVN (t(9)=–0.80; n.s.) and SON (t(9)=0.98; n.s) (Table 1). In contrast, POMC gene expression levels in the ARC were significantly reduced following one week of d-FEN treatment (t(9)=2.93; P=0.03).
The unaffected plasma ACTH (t(12)=–2.17; P=0.07), plasma corticosterone (t(12)=1.51; n.s) and relative adrenal weights (t(12)=–1.91; n.s) indicated unchanged activity of the HPA axis (Table 1).
d-Fenfluramine treatment in ABA rats
Since an effective dose of d-FEN was determined in ad libitum fed rats, this dose was used in ABA rats in order to influence the development and propagation of ABA. All rats developed hyperactivity following introduction of scheduled feeding. Substantial RWA was observed at the end of the light phase (ZT9-ZT12), which is referred to as food-anticipatory activity (FAA). However, FAA was not affected by d-FEN treatment (day F(6,84)=15.76; P < 0.001, day x treatment F(6,84)= 0.27; n.s). Total daily RWA (day F(6,84)=32.34; P < 0.001, day x treatment F(6,84)=0.51; n.s) was also not influenced by d-FEN treatment. In addition, cumulative FAA (day 0–6) (t(14)=1.26; n.s) and cumulative daily RWA (day 0–6) (t(14)=0.99; n.s) were not altered by d-FEN treatment (Figure 2A+B). Daily LMA, as measured by telemetric recordings, was not affected by d-FEN treatment over time (day F(6,78)=11.66; P < 0.001, F(6,78)=0.20; n.s), nor was daily cumulative LMA (day 0–6) (t(13)=0.48; n.s) (data not shown).
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Similar to food intake and RWA, relative body weight was unaffected following one week of d-FEN treatment (day F(6,84)=383.35; P < 0.001, day x treatment F(6,84)= 1.55; n.s) (Figure 2D). The final relative body weight of d-FEN-treated ABA rats was not different from vehicle-treated ABA rats (t(14)=0.90; n.s) and plasma leptin levels were below detection limits ( < 0.5 ng/ml) in both ABA groups.
The disbalance between energy intake and expenditure in ABA rats resulted in severe hypothermia. However, basal body temperature, measured during inactivity in the early light phase (ZT0-ZT3), was not affected by one week of d-FEN treatment (day F(6,84)=28.35; P < 0.001), day x treatment F(6,84)= 0.27; n.s) (Figure 2E).
Activation of the HPA-axis, as frequently seen in ABA rats, was not affected by d-FEN treatment. Plasma ACTH levels (t(13)=0.62; n.s.), plasma corticosterone (t(13)=0.87; n.s.) levels and relative adrenal weight (t(14)=0.49; n.s.) were not different between d-FEN-treated rats and vehicle-treated rats following one week of treatment (Table 2).
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). This corresponded with increased plasma osmolality following one week of d-FEN treatment in ABA rats (t(13)=–2.27; P=0.04) (Table 2). In addition, gene expression levels of AVP were increased in the SON (t(9)=–2.30; P=0.04) and in the PVN (t(9)=–2.71; P=0.03) following one week of d-FEN treatment. In contrast, POMC gene expression in the ARC was not affected by d-FEN treatment (t(12)=1.23; n.s.) (Table 2). |
Discussion |
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The absence of hypophagia in d-FEN-treated ABA rats was unexpected, since it was reported earlier that (one week of) d-FEN treatment reduced food intake in rats (Vickers et al. 2000, Rieg et al. 1994). This discrepancy might be explained by differences in experimental setup of the current and previous studies, such as duration of food access (1 h vs 1.5 h), running wheel access during feeding (access vs no access), gender (female vs male), age of the rats (±12 weeks vs 9 weeks) and strain of the rats (Wistar vs Sprague Dawley). Moreover, the experimental approach of the two ABA studies was different; while we examined the effects of d-FEN treatment on development and propagation of ABA during one week of treatment, Rieg et al.(1994) focussed the analysis on the day when 25% body weight loss was reached.
Whilst our study was being completed, another study was published, which showed that daily injections of d-FEN enhanced ABA (Atchley & Eckel 2005). This was caused by decreased food intake as well as by additional effects on energy expenditure, most likely being increased brown adipose tissue activity and increased lipid oxidation. In the present experiment, we found no evidence for anorexic effects of d-FEN in ABA rats. However, d-FEN-treated ad libitum fed rats reduced food intake showing efficacy of the dose (10 mg/kg/day) and treatment method (osmotic minipumps) used. Hence, the present results suggest that d-FEN-induced appetite suppression is overruled by physiological signals 0of starvation in ABA rats. Differences between the current study and the study of Atchley & Eckel (2005) might be explained by differences in experimental setup, such as the method of administration of d-FEN (osmotic minipump vs daily injections), dosage (10 mg/kg/day vs 0.5 mg/kg/day), duration of food access (1 h vs 2 h), strain of the rats (Wistar vs Long Evans) and possibly the stage of the oestrous cycle at the start of treatment. In an additional experiment, we treated ABA rats with a lower dose of d-FEN (1 mg/kg/day by osmotic minipump) for one week. However, we found that this dose of d-FEN did not affect any parameter of ABA and also did not reduce food intake in ad libitum fed rats (data not shown).
In the present study, we observed no effect of chronic d-FEN treatment on RWA or LMA in ABA rats. Total daily activity, activity during the dark phase (data not shown) as well as FAA, were not affected by d-FEN treatment. This corresponds with the two earlier studies, although in these studies total RWA was examined, and not specifically FAA (Rieg et al. 1994, Atchley & Eckel 2005). Moreover, effects of d-FEN on RWA, especially on FAA, could have been easily missed by Atchley & Eckel (2005) due to the timing of the d-FEN injections. Previously others reported reduced LMA and RWA in rats treated with d-FEN or other 5-HT2C preferring agonists during positive and negative energy balance (Aulakh et al. 1988, Kennett & Curzon 1988, Wilckens et al. 1992). Chronic fluoxetine (a SSRI) treatment also reduced RWA, whereas chronic parachlorophenylalanine (a tryptophane hydroxylase inhibitor) treatment increased RWA in semi-starved (SIH) rats (Altemus et al. 1996). From the aforementioned, it could thus be hypothesized that d-FEN treatment reduces wheel running in ABA rats. However, d-FEN treatment activates 5-HT2C receptors, that are located on POMC neurons (Heisler et al. 2002) and we showed before that chronic -MSH infusion increased wheel running, specifically FAA, in ABA rats (Hillebrand et al. 2005b). These data suggest that d-FEN treatment would increase wheel running in ABA rats. Altogether, unaffected wheel running following d-FEN treatment in the present as well as previous ABA experiments suggest the existence of compensating mechanisms.
Activation of the MC system following d-FEN treatment could not be confirmed. D-FEN treatment did not lead to a reduction in food intake or increased FAA as was observed following -MSH treatment. Moreover, d-FEN treatment did not increase (but tended to decrease) POMC mRNA expression after one week of treatment. Absence of early effects of chronic d-FEN treatment on development of ABA might be explained by already over-active POMC neurons during early days of ABA. We recently found transient up-regulation of POMC gene expression during early days of ABA (Hillebrand et al. 2005c. Following a few days of food restriction, compensating signals associated with severe negative energy balance could possibly override the putative d-FEN-induced stimulation of POMC neurons. In fact, in ad libitum fed rats a significant down-regulation of POMC was observed, which can be explained as a counter-regulatory response to a decrease in food intake and body weight. From this study it thus becomes clear that effects of acute and chronic treatment of d-FEN on e.g. POMC expression levels and food intake are not easily comparable (Choi et al. 2002, Heisler et al. 2002). Further studies should therefore be aimed at further analyzing actual 5-HT levels and 5-HT2CR binding on POMC neurons during development of ABA following chronic d-FEN treatment.
In the present study, water intake was measured to investigate specificity of d-FEN-induced effects on ingestive behaviour. Total daily water intake was reduced in ABA rats during the whole treatment period. As a consequence, plasma osmolality and mRNA levels of AVP, a neuropeptide involved in regulation of extra cellular fluid balance, were increased after 1 week of treatment. Ad libitum fed rats also showed a reduction of water intake following d-FEN treatment. However, this reduction was smaller than observed in ABA rats, and plasma osmolality and AVP gene expression were not changed following 1 week of treatment. Since rats often combine food and water intake, the reduction of water intake in ABA rats may be exaggerated as compared with ad libitum fed rats which eat more.
Strong and long-lasting effects of d-FEN treatment on water intake have not been reported before. A modest decrease in water intake was observed following acute injections of d-FEN, and during early days of long-term d-FEN treatment (Fletcher 1988, Roth & Rowland 1998, Vickers et al. 2003), and seems to be mediated by 5-HT2C or 5-HT1B receptors (Clifton et al. 2000, Lee et al. 2002). The MC system seems not to be involved in the effect of d-FEN on water intake; chronic intracerebroventricular (icv) -MSH treatment does not lead to reduced water intake of ABA rats (J J G Hillebrand, M J H Kas, R A H Adan, 2005).
D-FEN-induced changes in AVP gene expression have not been described before, whereas d-FEN-induced increases in plasma AVP levels are generally known (Iovino & Steardo 1985, Max 1994, Saydoff et al. 1996). Regarding plasma hyperosmolality (van Tol et al. 1987, Amaya et al. 1999) in ABA rats and the only modest activation of c-fos in AVP neurons following d-FEN treatment (Javed et al. 1999, Mikkelsen et al. 1999), the up-regulation of AVP expression is probably an indirect result of hypodypsia and not of a direct effect of d-FEN on AVP neurons. Nevertheless there are also indications that plasma AVP levels are at least partly mediated via 5-HT2CR and the angiotensin system (Saydoff et al. 1996). Increased AVP release by the pituitary following acute d-FEN or 5-HT treatment can be reduced by inhibiting angiotensin II production or by antagonizing angiotensin 1 or 5-HT2AC receptors. In the present experiment we did not measure AVP release (but central AVP expression) and used chronic d-FEN treatment instead of acute treatment (which is not easily comparable), therefore, we consider increased AVP expression as a secondary response to hypodipsia, which is observed to a greater extent in ABA rats than in ad libitum fed rats.
We observed no changes in 1 h food intake, RWA, body weight loss or HPA axis activation in d-FEN-treated ABA rats. Strikingly, these three important parameters of ABA are influenced by chronic -MSH treatment. In addition, POMC mRNA expression levels in ABA rats were not increased (but tended to be decreased) following one week of d-FEN treatment. A prolonged decrease in water intake was observed in d-FEN-treated ABA rats, which corresponded with increased plasma osmolality and increased AVP gene expression. d-FEN was an effective appetite reducing agent for treating obesity. However, it was withdrawn from the market in 1997 because of potential harmful side effects, such as pulmonary hypertension and unusual valvular morphology and regurgitation (Guy-Grand 1995, Connolly et al. 1997, MacLean 1999). Yet no information is available on its effects on fluid intake, although patients often complained of a dry mouth and drowsiness (Connolly et al. 1997, Bray & Greenway 1999). We conclude that d-FEN treatment results in a strong and prolonged reduction of water intake in food-restricted running rats, but does not enhance the development and propagation of ABA in this study.
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Acknowledgements |
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Received in final form 11 November 2005
Accepted 21 November 2005
Made available online as an Accepted Preprint 24 November 2005
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