Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 492141, 8 pages http://dx.doi.org/10.1155/2014/492141 Research Article Myristic Acid Produces Anxiolytic-Like Effects in Wistar Rats in the Elevated Plus Maze Carlos M. Contreras,1,2 Juan Francisco Rodríguez-Landa,2 Rosa Isela García-Ríos,2 Jonathan Cueto-Escobedo,2 Gabriel Guillen-Ruiz,2 and Blandina Bernal-Morales2 1 Unidad Periferica´ Xalapa, Instituto de Investigaciones Biomedicas,´ Universidad Nacional Autonoma´ de Mexico,´ 91190 Xalapa, VER, Mexico 2 Laboratorio de Neurofarmacolog´ıa, Instituto de Neuroetolog´ıa, Universidad Veracruzana, Avenida Dr. Luis Castelazo s/n, Colonia Industrial Las Animas,91190Xalapa,VER,Mexico´ Correspondence should be addressed to Carlos M. Contreras; [email protected] Received 17 July 2014; Revised 1 September 2014; Accepted 4 September 2014; Published 18 September 2014 Academic Editor: Saulius Butenas Copyright © 2014 Carlos M. Contreras et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A mixture of eight fatty acids (linoleic, palmitic, stearic, myristic, elaidic, lauric, oleic, and palmitoleic acids) at similar concentrations identified in human amniotic fluid produces anxiolytic-like effects comparable to diazepam in Wistar rats. However, individual effects of each fatty acid remain unexplored. In Wistar rats, we evaluated the separate action of each fatty acidat the corresponding concentrations previously found in human amniotic fluid on anxiety-like behaviour. Individual effects were compared with vehicle, an artificial mixture of the same eight fatty acids, and a reference anxiolytic drug (diazepam, 2 mg/kg). Myristic acid, the fatty acid mixture, and diazepam increased the time spent in the open arms of the elevated plus maze and reduced the anxiety index compared with vehicle, without altering general locomotor activity. The other fatty acids had no effect on anxiety- like behaviour, but oleic acid reduced locomotor activity. Additionally, myristic acid produced anxiolytic-like effects only when the concentration corresponded to the one identified in human amniotic fluid (30 g/mL) but did not alter locomotor activity. We conclude that of the eight fatty acids contained in the fatty acid mixture, only myristic acid produces anxiolytic-like effects when administered individually at a similar concentration detected in human amniotic fluid. 1. Introduction that were comparable to diazepam in male and female adult Wistar rats [7, 8]. This fatty acid mixture, based on the Odors from amniotic fluid produce calming reactions and content and concentration in human amniotic fluid, included attraction in human newborns [1] and also newborns from C12:0 (lauric acid), C14:0 (myristic acid), C16:0 (palmitic other species [2–4], suggesting specific effects of amniotic acid), C16:1 (palmitoleic acid), C18:0 (stearic acid), C18:1 cis fluid on emotion and anxiety [5]. An artificial mixture of six (oleic acid), C18:1 trans (elaidic acid), and C18:2 (linoleic fatty acids (capric acid, lauric acid, myristic acid, palmitic acid) and produced appetitive responses in human newborns acid, oleic acid, and linoleic acid) identified in pig amniotic [9].However,unknowniswhichoftheeightfattyacids fluid, colostrum, and maternal milk (Sus scrofa,LargeWhite contained in human amniotic fluid is mainly responsible for breed) exerts appeasing actions in piglets [6]. An anecdotal its anxiolytic-like effect. report suggested that the same artificial mixture also exerted Thepresentstudycomparedtheeffectsofeachofthe anxiolytic-like effects in children [6]. eight fatty acids against an artificial fatty acid mixture that In human amniotic fluid, eight fatty acids from colostrum contained the same eight fatty acids, with diazepam as a and maternal milk were identified, and an artificial mixture reference anxiolytic drug, in Wistar rats subjected to the of such human fatty acids produced anxiolytic-like effects elevated plus maze [10]. We identified the most potent fatty 2 BioMed Research International acid among those contained in the fatty acid mixture in Cohen et al. [14] as follows: Anxiety Index = 1 − [([Open arm producing changes in the elevated plus maze and tested three time/Test duration] + [Open arms entries/Total number of different concentrations of this fatty acid in the same test. entries])/2]. 2. Materials and Methods 2.4.2. Locomotor Activity Test. To evaluate the effects of the treatments on spontaneous locomotor activity and discard 2.1. Ethics. Alloftheratproceduresfollowedtheprinciples the possibility of hypoactivity or hyperactivity attributable ofanimalcarebasedontheGuide for the Care and Use to the treatments that could influence performance in the of Laboratory Animals [11]andNorma Oficial Mexicana elevated plus maze, a 5 min locomotor activity test was para el Cuidado y Uso de Animales de Laboratorio (NOM- performed after the elevated plus maze test in another room 062-ZOO-1999) [12]. The protocol received authorization (40 lux). We used an automated locomotor activity moni- from the Biomedical Research Institute Ethical Committee tor (Acti-Track v2.7.10, PanLab, S.L., Instrument, Barcelona, (Universidad Nacional Autonoma´ de Mexico).´ Spain) constructed of a Perspex box (45 cm × 45 cm) with 35 cm high walls. The apparatus was elevated 3 cm above the box frame floor. A total of 32 infrared beams, 16 on 2.2. Preparation of Fatty Acid Mixture. The fatty acid mixture each perpendicular wall, were connected to an interface (LE was prepared according to previous studies that reported 8811, LSI Letica Scientific Instrument, Barcelona, Spain) and its anxiolytic-like effects7 [ , 8], based on the concentrations subsequently to a computer. For data analysis, the floor of the found in human amniotic fluid. The mixture consisted of cage was virtually divided into five zones (four peripheral and linoleic acid (4.4 mg), palmitoleic acid (7.1 mg), stearic acid onecentral),andwemeasuredthenumberofentriesinto (3.7 mg), myristic acid (3.0 mg), elaidic acid (1.5 mg), lauric each zone (crossings), total resting time, and total activity acid (0.4 mg), oleic acid (8.0 mg), and palmitic acid (15.3 mg) time during the 5 min test as indicators of locomotion. No dissolved in 100 mL of vehicle (96% propylene glycol and ∘ other behaviors, such as rearing or grooming, were evaluated. 4% ethanol) at a temperature <40 C. Each fatty acid that Because of the relatively small cage, we did not compare was contained in the mixture was also prepared in the same central versus peripheral exploration. quantity and individually dissolved in a volume of 100 mL of After each experimental session, the elevated plus maze vehicle, maintaining the same concentration found in human andlocomotoractivityboxwerecarefullycleanedand amniotic fluid. All of the fatty acids were of analytical grade deodorized with a 5% ethanol cleaning solution. Approxi- and purchased from Sigma-Aldrich (St. Louis, MO, USA). mately 5 min elapsed between each test to allow the scent of Propylene glycol and ethanol were obtained from J. T. Baker thesubstancestodissipate. (Xalostoc, MEX, Mexico). 2.5. Experimental Groups and Treatments. The rats were 2.3. Animals. The experiment included 149 male Wistar rats assigned to 11 experimental groups: (i) vehicle (=10), obtained from a local strain supplied by Harlan (Mexico City, (ii) fatty acid mixture (FAT-M; =9), (iii) linoleic acid Mexico). The rats were maintained in local housing facilities =10 = ∘ (44 g/rat, ), (iv) palmitoleic acid (71 g/rat, at a mean temperature of 25 C with a 12 h/12 h light/dark 10), (v) stearic acid (37 g/rat, =10), (vi) myristic acid cycle (lights on at 7:00 a.m.). All of the rats included in the (30 g/rat, =10), (vii) elaidic acid (15 g/rat, =10), (viii) study were 11-12 weeks old, weighed 200–250 g, and were lauric acid (4 g/rat, =10), (ix) oleic acid (80 g/rat, = × housed five rats per cage in acrylic boxes (44 cm width 10), (x) palmitic acid (153 g/rat, =10), and (xi) diazepam × 33 cm length 20 cm height) with ad libitum access to food (2 mg/kg, =10). All of the injections were administered (Teklad lab animal diets, Harlan, Indianapolis, IN, USA) and subcutaneously in a volume of 1 mL/rat, with the exception purified water. of diazepam, which was administered intraperitoneally. The rats were first tested in the elevated plus maze (5 min) and 2.4. Behavioral Tests then in the locomotor activity test (5 min). One hour elapsed between the injections of substances and the beginning of the 2.4.1. Elevated Plus Maze. The elevated plus maze was con- behavioral tests. structedofwoodandsituatedinaroomilluminatedat40lux. In another group of rats, we evaluated three concentra- The apparatus consisted of two opposite open and closed tions of the fatty acid that produced significant anxiety-like arms set in a plus configuration. The dimensions of the open behavior in the elevated plus maze. Four experimental groups arms were 50 cm × 10 cm (length × width) and the closed were included: vehicle (=10), one-third the concentration arms were 50 cm × 10 cm × 40 cm (length × width × height). of the fatty acid found in human amniotic fluid=10 ( ), the The entire maze was elevated 50 cm from the floor. The rats full concentration of the fatty acid found in amniotic fluid were individually placed at the center of the maze, facing an (=10), and double the concentration of the fatty acid open arm, and the time spent on and number of entries into found in human amniotic fluid=10 ( ). All of the injections theopenarmswererecorded[13]. The total number of entries were administered subcutaneously in a volume of 1 mL/rat, (open arms + closed arms) and percentage of open arm 1 h before the behavioral tests. The rats were tested first in the entries ([open entries]/[total entries] × 100) were calculated. elevated plus maze (5 min) and then in the locomotor activity Additionally, the anxiety index was calculated according to test (5 min).