Prenatal Exposure to Fenugreek Impairs Sensorimotor Development and the Operation of Spinal Cord Networks in Mice Loubna Khalki1,2, Saadia Ba M’hamed1, Zahra Sokar1, Mohamed Bennis1, Laurent Vinay2, Hélène Bras2☯, Jean-Charles Viemari2*☯ 1 Laboratoire Pharmacologie, Neurobiologie et Comportement, Centre National de la Recherche Scientifiques et Techniques (URAC 37), Cadi Ayyad Université, Marrakech, Maroc, 2 Institut de Neurosciences de la Timone, P3M Team, CNRS, Aix Marseille Université, Marseille, France
Abstract Fenugreek is a medicinal plant whose seeds are widely used in traditional medicine, mainly for its laxative, galactagogue and antidiabetic effects. However, consumption of fenugreek seeds during pregnancy has been associated with a range of congenital malformations, including hydrocephalus, anencephaly and spina bifida in humans. The present study was conducted to evaluate the effects of prenatal treatment of fenugreek seeds on the development of sensorimotor functions from birth to young adults. Pregnant mice were treated by gavage with 1g/kg/day of lyophilized fenugreek seeds aqueous extract (FSAE) or distilled water during the gestational period. Behavioral tests revealed in prenatally treated mice a significant delay in righting, cliff avoidance, negative geotaxis responses and the swimming development. In addition, extracellular recording of motor output in spinal cord isolated from neonatal mice showed that the frequency of spontaneous activity and fictive locomotion was reduced in FSAEexposed mice. On the other hand, the cross-correlation coefficient in control mice was significantly more negative than in treated animals indicating that alternating patterns are deteriorated in FSAE-treated animals. At advanced age, prenatally treated mice displayed altered locomotor coordination in the rotarod test and also changes in static and dynamic parameters assessed by the CatWalk automated gait analysis system. We conclude that FSAE impairs sensorimotor and coordination functions not only in neonates but also in adult mice. Moreover, spinal neuronal networks are less excitable in prenatally FSAE-exposed mice suggesting that modifications within the central nervous system are responsible, at least in part, for the motor impairments. Citation: Khalki L, Ba M’hamed S, Sokar Z, Bennis M, Vinay L, et al. (2013) Prenatal Exposure to Fenugreek Impairs Sensorimotor Development and the Operation of Spinal Cord Networks in Mice. PLoS ONE 8(11): e80013. doi:10.1371/journal.pone.0080013 Editor: Thomas H Gillingwater, University of Edinburgh, United Kingdom Received May 30, 2013; Accepted September 27, 2013; Published November 5, 2013 Copyright: © 2013 Khalki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors acknowledge support from the Groupement De Recherche International en Neuroscience GDRI-Neuro (CNRS/CNRST/INSERM) and the NEUROMED project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail:
[email protected] ☯ These authors contributed equally to this work.
Introduction
developmental toxicity [7-9]. Furthermore, we previously demonstrated that prenatal exposure to fenugreek seeds produced neurobehavioral alteration at P21 including decreased locomotor activity, impaired motor coordination and spatial short term memory [10]. Because of the widespread use of fenugreek seeds for its therapeutic actions during pregnancy, it is important to study the effects of FSAE on the development of neuronal networks such as the spinal locomotor network. Our aim was to investigate short and long term effects of FSAE exposure during gestation on locomotor functions, by using both in vitro and in vivo experimental paradigms. During the first postnatal week, spontaneous locomotor activity consists of crawling movements [11]. During this period, the hindlimbs remain passive. These movements
Fenugreek (Trigonella foenum graecum) is one of the oldest traditional medicinal plants, cultivated in India, the Mediterranean region, North Africa and Yemen (Kassem et al., 2006). Fenugreek seeds are commonly used worldwide for its laxative and galactagogue properties but also to stimulate appetite and prevent stomach ache [1]. More recently, immunostimulatory, antidiabetic, antihypertensive and cholesterol-lowering potentials have been described [2-4]. However, consumption of fenugreek seeds during pregnancy has been associated with congenital malformations, neonatal and birth defects [5,6]. Oral treatment with the fenugreek seeds aqueous extract (FSAE) also causes reproductive and
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(HED) from animal data [22]. Translation of our data from mice weighting 25g to humans with a weight of 70 kg is given by the following equation: HED = Animal Dose (mg/kg) x (animal wt/human wt in kg)0.33 = (1000 mg/kg) x (0.025 kg/70kg)0.33 = 72.85 mg/kg = 5.1 g/70 kg This daily dose of 5.1 g appears to be on the very low side of human consumption.
are gradually replaced by crawling with all four limbs and around postnatal day 10, by walking on all fours with the belly free from the floor [12,13]. In spinal cord, a spontaneous bursting activity can be recorded in vitro from ventral roots during the perinatal period [14-16]. In vitro, a fictive locomotor pattern can also be elicited following bath application of excitatory amino acid agonists and monoamines such as serotonin (5HT) [17] or electrical stimulation of some brainstem areas [18-21]. At early postnatal days (P0-P3), we investigated whether spontaneous and locomotor-like activities were altered after prenatal exposure to FSAE. Then, at a later developmental period (P5-P12) we tested the pup‘s reflexes and swimming performance as indicators of the sensorimotor development. Finally, we assessed static and dynamic changes in juvenile (P21) and young adults (P41) using the CatWalk analysis.
Evaluation of short term effects of prenatal exposure to fenugreek We investigated the righting reflex and the cliff avoidance during the first postnatal week. and the geotaxis response at P10 and P12 [23]. Surface righting reflex (P5, P7 and P9): this test evaluates motor function and coordination. Each pup was placed on its back on a flat surface and released. The time required to get back on all four paws was measured. The maximum time allotted to each trial was 30 seconds. Cliff avoidance (P6): the pup was placed on a table edge with the forepaws and nose over the edge. The time taken to make a U- turn was noted. Each pup was tested once. The maximum time allowed per trial was 60 seconds. Negative geotaxis (P10 and P12): coordination can be examined using the negative geotaxis test, an automatic, stimulus-bound orientation movement which provides informations about vestibular and/or proprioceptive functions. The time taken to make a U-turn from a head-down position on a 35-degrees inclined plywood surface was measured. This test has been performed after P8 because it requires crawling movements that appear at the end of the first postnatal week [23]. Swimming development: as newborn mice are able to swim straight at P8 [23], we started to analyse swimming at this age and repeated the same analysis at P10 and P12. Each mouse was placed in a water tank (28°C) for 5–10s. The direction, angle in the water (head position) and limb involvement were observed and scored. Direction was scored as follows: sinking: 0; floating : 1; circling : 2; swimming straight or nearly straight : 3. Angle scores consisted of head submerged : 0; nose at the surface : 1; nose and top of head at or above the surface but ears still below the surface: 2; ears half way above the surface : 3; ears completely above the surface : 4. The involvement of limbs was evaluated by the following scores : no paddling, 0; paddling with all four limbs : 1; paddling with only the hind limbs and the forelimbs remaining motionless : 2. The experimenters conducting the behavioral tests were blinded to the animal condition.
Material and Methods Ethics statement The experimental procedures conformed to the guidelines of the University of Marrakech and the European (Council Directive 86/6009/EEC) and French regulations (Ministry for Agriculture and Fisheries, Division of Animal Rights). Experimental procedures were approved by the Institut de Neuroscience de la Timone Ethics Committee registered at the National Commission of animal experimentation (autorized No. 71). Efforts were made to minimize the number of animals used. Adequate measures were taken to minimize pain and animal discomfort.
Preparation of extract Trigonella foenum-graecum seeds were collected in the area of Settat, Morocco. The plant material was identified in the Department of Biology, Faculty of Sciences Semlalia, Cadi Ayyad University. A voucher specimen (n = 5511) was deposited at the herbarium of the mentioned faculty. The aqueous extract of fenugreek (Trigonella foenum-graecum) was obtained by agitating powder obtained from seeds in distilled water (1g/20 ml distilled water). The extract obtained was centrifuged and the supernatant was lyophilized (final yield of 20%) and stored at -20°C until further use.
Animals and treatment Swiss CD1 mice (25–30 g) were housed in groups of 4-5 with a 12 h light ⁄ dark cycle. Food and water were available ad libitum in the home cages. Males were housed overnight with females in proestrus stage (1: 2), and the females certified to be pregnant were divided into two groups of 17 animals. The first group used as controls received distilled water, while the second group was treated with aqueous extract of fenugreek (Trigonella foenum-graecum). Mice were treated by gavage with 1g/kg/day of lyophilized FSAE during the gestational period [9,10]. Doses up to 100 g/day, are reported to be taken by pregnant women [5]. The American Federal Drug Administration (FDA) Guidance for Industry (2005) provided an equation that enables to identify the human equivalent dose
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Electrophysiological recordings Mice (post-natal day P0- P 3) were deeply anesthetized by hypothermia. After decapitation, the lumbar spinal cord was dissected in an artificial cerebrospinal fluid of the following composition (in mM): 128 NaCl, 4 KCl, 1.5 CaCl2, 1 MgSO4, 0.5 NaH2PO4, 21 NaHCO3 and 30 glucose; oxygenated with 95% O2 and 5% CO2, pH 7.4. The spinal cord and roots were removed from sacral segments up to T8-T10. The preparation was pinned down, ventral side up, in the recording chamber, and continuously perfused with the same saline solution as the
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Statistical analysis
one used for dissection. The temperature was set to 28–30°C. Spontaneous and locomotor-like activities were recorded from lumbar ventral roots (left ⁄ right L2 and L5) by means of glass suction electrodes connected to an AC-coupled amplifier (bandwidth: 70 Hz to 3 kHz). Fictive locomotion was elicited by a bath application of N-methyl-DL-aspartic acid (NMA) ⁄ 5hydroxytryptamine creatinine sulfate (5-HT) for 30min (NMA, 10 µM; 5-HT, 10 µM). All compounds used were purchased from Sigma. Electrophysiological data were acquired through a Digidata 1440A interface using the clampex 10 software (Molecular Devices, Sunnyvale, CA, USA). Data analysis consisted of rectifying and integrating (time constant 50 ms) the raw extracellular recordings from ventral roots. The phase relationships were evaluated on 10 successive 60-s recordings by means of cross-correlation analysis. The locomotor-related coordinations were assessed by the cross-correlation coefficient at zero phase lag (center of the cross-correlogram). The mean correlation coefficient (R) is the average of r values collected from all animals a given experimental condition. Bursts were automatically detected with clampfit 10.0 software (Molecular Devices). After setting a Y-threshold value in the recording, the program detects events above this value”. We then used the “analysis event” function and then the “burst analysis” tool that calculates the mean duration and the frequency of bursts [16].
Data were analyzed using Prism 4 software (GraphPad) and are presented as mean and standard errors. Differences between groups were tested by ANOVA 2 variance and the Mann Whitney non parametric test. Chi- square test was used to analyze the step patterns in the gait analysis. Linear regression was also used to assess whether the time required for the righting reflex was correlated with postnatal age. All values are presented as mean ± sem. The level of significance was p ≤ 0.05.
Results Short (P0-P12) and long term effects (P21-P42) of prenatal FSAE exposure on the locomotor activity were investigated using electrophysiological recordings, behavioral tests and gait analysis.
Prenatal exposure to FSAE affected the sensorimotor development We tested the influence of FSAE exposure on sensorimotor tests performed during the postnatal period to evaluate the proper development of the sensorimotor capacities. First, we studied the surface righting reflex that requires the integrity of muscular and motor functions as well as labyrinthine ones. The time required for the surface righting response significantly decreased with age in controls (Spearman Test, P = 0.0049) whereas it did not significantly change in FSAE treated mice (Spearman Test, P = 0.15). Moreover, the righting response was significantly longer in FSAE exposed pups compared with controls at P7 (23.3 ± 3.1 s vs 9.4 ± 3.4 s, (t = 3.7; P< 0.01) and P9 (12.4 ± 3.7 s vs 1.7 ± 0.3 s, t = 2.9, P < 0.05; Figure 1A). Second, we evaluated the geotaxis reflex. When placed on a 35° angle slope, most control mice turned around to orient their body with the head upward within 4s (Figure 1B). This negative geotaxis response was much slower in treated mice as reflected by a ~300% increase in time at both ages tested [P10 (t = 3.9; P < 0.01) and P12 (t = 3.1; P < 0.05)]. Third, we performed the cliff avoidance test: the time taken by treated animals to turn and crawl away from the cliff drop was significantly increased compared with controls (20.8 ± 2.9 s vs 11.9 ± 1.2 s in control; P < 0.01; Figure 1C). We also analyzed the swimming development, with regard to the swimming direction, the treated group had a lower score than the controls at P8 (t = 9; P
