Effects of 17 b-estradiol and extremely low-frequency electromagnetic fields on social recognition memory in female rats: A possible interaction?

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Effects of 17 b-estradiol and extremely low-frequencyelectromagnetic fields on social recognition memoryin female rats: A possible interaction?

Gloria Reyes-Guerrero a, Mario Vázquez-García a,⁎, David Elias-Viñas b,Olga A. Donatti-Albarrán a, Rosalinda Guevara-Guzmán a

aDepartamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México,Apdo. Postal 70250, México, D. F., 04510, MéxicobDepartamento de Ingeniería Eléctrica, Sección de Bioelectrónica, Centro de Investigación y de Estudios Avanzados,IPN, México, D.F. 07360, México

A R T I C L E I N F O

⁎ Corresponding author. Fax: +52 01 525 63528E-mail address: [email protected]

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.04.020

A B S T R A C T

Article history:Accepted 4 April 2006Available online 30 May 2006

We have investigated a potential memory-enhancing effect of exposure to extremely low-frequency electromagnetic fields (ELF EMF) in female rats and its dependence on estrogen,using a social recognition task. A juvenile social recognition paradigm was used andmemory retention tested at 30 and 300 min after an adult was exposed to a juvenileduring two 4-min trials. Results showed that an intact social recognition memory waspresent at 30 min in both gonadally intact and ovariectomized rats with, or without, ELF-EMF. However, whereas gonadally intact control females failed to show retention of therecognition memory at 300 min, those additionally exposed to ELF EMF did. This showsthat the enhanced duration effect of ELF EMF on social recognition memory occurs ingonadally intact females as well as in males. In addition, results showed that the ELF EMFfacilitation of memory retention was prevented by ovariectomy but restored byexogenous treatment with estrogen. This suggests that this ELF EMF effect on socialrecognition memory is estrogen-dependent.

© 2006 Elsevier B.V. All rights reserved.

Keywords:EstrogenSocial behaviorOlfactory memoryShort-term memoryLearningElectromagnetic field

1. Introduction

Evidence from neuroanatomical, electrophysiological andbehavioral studies supports a role for magnetic fields inlearning and memory processes. Most of what is knownabout the functional impact of magnetic fields on the centralnervous system is derived from studies of neurotransmitterrelease (Pesic et al., 2004; Zecca et al., 1991) and neuraldisorders (Poirrier et al., 2004) and behavioral studies (Lai,2004; Saunders, 2005). Over the past few decades, emission of

90.d.unam.mx (M. Vázquez-

er B.V. All rights reserved

EMF by electronic devices and other systems has becomerelevant for its effects on neural mechanisms in humans andsome vertebrates. Exposure to extremely low-frequencyelectromagnetic fields (ELF EMF) has apparently oppositeeffects on learning and memory processes probably affectingneurotransmitter function (Kavaliers et al., 1993; Kavaliers etal., 1996; Lai, 2004; Prato et al., 1995; Zecca et al., 1991). Thus,acquisition from behavioral task is modified by ELF EMF(Kavaliers et al., 1993; Kavaliers et al., 1996; Sienkiewicz et al.,1998; Sienkiewics et al., 2001).

García).

.

Fig. 1 – Mean investigation time (x ± SEM) during first(solid bars) and second (grey bars) encounters of socialrecognition responses of adult female rats tested at 30-minIEI. Non-ELF EMF-stimulated animals (A, left panel) and ELFEMF-stimulated animals (A, right panel) show significantdifferences between first and second encounters, t test,P = 0.0008 and P = 0.002, respectively, n = 8. In panel (B) theovariectomized non-ELF EMF-stimulated rats (left panel) andthe ovariectomized plus ELF EMF-stimulated rats (rightpanel), also show significant differences between bothencounters, respectively. t tests, P = 0.001 and P = 0.004respectively.

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We have recently demonstrated using a social recognitiontask that exposure to ELF EMF extended social memoryduration to 300 min in adult male rats (Vázquez-García et al.,2004). However, the existence of recognized sex differencesunderlying mechanisms of social recognition (Ferguson etal., 2002) makes it important to evaluate the effects of ELFEMF on this social memory in female as well as in male rats.

Ovarian hormones have been shown to exert a powerfulinfluence on learning and memory processes (Frye, 1995;Galea et al., 2001; O'Neal et al., 1996; Sánchez-Andrade etal., 2005; Tanabe et al., 2004; Warren and Juraska, 1997).Acquisition of spatial learning tasks appears to be impairedfollowing replacement of estradiol (E2) in ovariectomizedvoles (Galea et al., 2002). Indeed, it has been reported thathigh levels of E2 in adult female rats are associated withimpaired and low levels of E2 with improved, spatiallearning (Frye, 1995; Galea et al., 1995; Galea et al., 2001;Holmes et al., 2002; Luine et al., 1998; Warren and Juraska,1997). However, Sandstrom and Williams (2001) reportedthat E2 increases excitatory connectivity in the hippocam-pus and improves spatial memory retention in ovariecto-mized rats. Likewise, Woolley and McEwen (1993) reported atransient increase in dendritic spine density of CA1pyramidal neurons that peaks approximately 48 h followinga second injection of E2 and which returns to baseline levelsover the course of a week. Also, estrogen replacementincreases the interest in social interaction in ovariectomizedmice (Tang et al., 2005).

The discrepancies in the literature concerning the effectof estradiol on cognition may reflect the differential involve-ment of brain regions recruited during the various types oftasks utilized. Consequently, E2 may differentially affectvarious cognitive processes and the corresponding brainregions that mediate behaviors associated with these.Performance in the Morris Water Maze task and the spatialworking-reference memory version of the radial arm mazeboth rely on the integrity of the hippocampus (Jarrard, 1993;Morris et al., 1982; O' Keefe et al., 1975). However, there isevidence that estradiol may affect other types of memorythat are mediated by brain regions independent of thehippocampal formation. For example, in ovariectomized ratstreated with E2, social recognition behavior that depends ofthe integrity of the olfactory bulbs was improved, suggestingthat estradiol plays an important role in social recognition(Hlinak, 1993). Several lines of evidence also suggest that E2mediates socially motivated behaviors in rodents (Imwalle etal., 2002). Mice lacking a fully functional estrogen receptor αor β showed reduced chemo-investigatory behavior (Choleriset al., 2003; Imwalle et al., 2002; Rissman et al., 1997;Rissman et al., 2002).

It has been shown that short-term social memory ismainly dependent upon olfactory cues (Dluzen et al., 1998;Ferguson et al., 2002; Sawyer et al., 1984; Thor and Holloway,1982; Young, 2002), and that sexual dimorphism may reflectdifferential dependence of the sexes on oxytocinergic(female) and vasopressinergic (male) inervation of limbicbrain areas (Engelmann et al., 1998; Ferguson et al., 2001;Ferguson et al., 2002). Furthermore, oxytocin effects infemale rats are mediated, al least in part, by estrogens(Choleris et al., 2003; Tang et al., 2005).

The aim of the present study was therefore to investigatewhether ELF EMF would also affect the duration of short-termsocial memory in female rats and whether this action wasestrogen-dependent.

2. Results

All 48 OVX-female rats were in anestrus (diestrous stage)based upon cytology from vaginal smears. In 16 out of the 48OVX-female rats, the effectiveness of estrogen replacementwas demonstrated by changes in the stage of estrous cycle,from diestrous to estrus.

Fig. 1 shows the mean duration in investigation at IEI of30 min by the 32 adult female rats tested. Fig. 1A depictsthe mean time spent in social investigation by the non-ELFEMF-stimulated group (left panel) and by the ELF EMF-stimulated group (right panel). In both the non-ELF EMF-stimulated group (t test, P = 0.0008, n = 8) and ELF-stimulated one (t test, P = 0.002, n = 8) the investigation

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time of the juvenile in the first encounter was significantlygreater than in the second. Fig. 1B shows that the meanduration of social investigation times were also significantlydifferent between first and second encounters in the OVX-non-ELF EMF-stimulated group (left panel) (t test, P = 0.001,n = 8) and in the OVX-ELF EMF-stimulated group (rightpanel) (t test, P = 0.004, n = 8). The mean duration ininvestigation time for the ELF EMF-stimulated group wasshorter than the mean for the non-ELF EMF-stimulated group,while there were no differences between the non-ELF EMF-stimulated group and OVX-non-ELF EMF-stimulated andOVX-ELF EMF-stimulated groups (ANOVA, F(3,28) = 3.63,P = 0.02, for the first encounter and, ANOVA, F(3,28) = 4.27,P = 0.03 for the second encounter). However, analysis of theratio index did not show differences between these groups ofanimals (Table 1).

Fig. 2 shows the mean duration of investigation whenthe IEI between two encounters was 300 min. The meaninvestigation times for both encounters measured in thenon-ELF EMF-stimulated group (n = 8) were not signifi-cantly different (t test, P = 0.2) (Fig. 2A, left panel).However, investigation times in the ELF EMF-stimulatedrats were decreased upon re-exposure to the samejuvenile indicating that the memory was still intact (ttest, P = 0.002) (Fig. 2A, right panel). The investigationtimes during second encounter were also significantlydifferent between the non-ELF EMF-stimulated and ELFEMF-stimulated groups (P = 0.001). Retention of socialrecognition memory at 300-min IEI is therefore improvedby ELF EMF stimulation.

In order to assess the possibility that the increasedduration of social recognition memory involves gonadalhormones, ELF EMF was applied in rats (n = 16) which hadbeen ovariectomized three weeks before and then tested inthe social recognition task. Fig. 2B (left panel) shows thatELF EMF failed to produce significant reductions in juvenileinvestigation times at 300 min (P = 0.81) in contrast togonadally intact animals (Fig. 2A, right panel). However, inthe group of ovariectomized animals treated with estradiol

Table 1 – Mean investigation time (x ± SEM) of adultfemale rats

Groups IEI (min) n Ratio index (x ± SEM)

Non-ELF EMF 30 8 0.55 ± 0.06ELF EMF 30 8 0.46 ± 0.07OVX non-ELF EMF 30 8 0.48 ± 0.09OVX-ELF EMF 30 8 0.56 ± 0.04Non-ELF EMF 300 8 1.30 ± 0.24ELF EMF 300 8 0.48 ± 0.06c

OVX-ELF EMF 300 8 1.09 ± 0.17OVX-ELF EMF + E2 300 8 0.53 ± 0.06c

OVX non-ELF EMF 300 8 1.05 ± 0.30OVX non-ELF EMF + E2 300 8 1.13 ± 0.25

Time spent (in seconds) by the adults in investigating conspecificjuveniles during each encounter.aP < 0.005 vs. first encounter and bP < 0.05 vs. first encounter (t test).Kruskal–Wallis test followed by Dunn's post hoc comparison testshowed a significant decrease of the ratio index. cP < 0.05 vs.corresponding group.

the increased retention effect (as evidenced by significantlyreduced investigation times at 300 min) seen in gonadallyintact individuals was restored (Fig. 2B) (t test, P = 0.02). AKruskal–Wallis ANOVA test also revealed a significantdifference in ratio index under these conditions (H = 14.4,P = 0.002) (Table 1). Post hoc analysis showed that bothintact ELF EMF-stimulated rats and OVX-ELF EMF-stimulatedand injected with E2 rats had a significantly decreasedinvestigation ratio index (P < 0.05), indicating that theseadult animals recognize the previously encountered juvenileas familiar after a 300-min IEI.

To control for the possibility that E2 treatment alonemight have caused the retention of the recognition memoryat 300 min, a further two groups of 8 OVX rats were testedwhich were either injected with E2 or an oil vehicle. Fig. 3shows that neither group showed significant differences ininvestigation times between the first and second encounters(P = 0.35 and P = 0.72, respectively. This suggests thatincreased memory retention seen after ELF EMF treatmentwas due to its combination with E2 treatment and not to E2alone.

Even though the mean investigation time in the firstencounter in control groups, i.e., non-ELF EMF-stimulatedrats at 30-min IEI (Fig. 1A, left panel) and non-ELF EMF-stimulated rats at 300-min IEI (Fig. 2A, left panel), was different(58.9 ± 5.72 s and 42.7 ± 5.90 s, respectively), they did not quiteachieve significance (t test, P = 0.056).

Table 1 summarized the mean investigation times andratio index at 30 and 300min from intact non-ELF EMF and ELFEMF experiments as well as OVX non-ELF EMF, OVX-ELF EMFand OVX-ELF EMF + E2 experiments.

To determine whether retention of social recognitionmemory varied across the estrous cycle, we analyzed 56out of all 80 adult female rats in both ELF EMF and nonELF EMF-stimulated conditions and including OVX rats(Table 2). Because mean investigation time measurebetween non-ELF EMF and ELF EMF at 30-min IEI and ELFEMF at 300-min IEI did not differ significantly in the first(P = 0.21) and second (P = 0.07) encounters, we combinedrats tested at 30-min IEI and rats at 300 min IEI into threegroups designated pro-estrus, estrus and diestrus. In 24rats with regular estrous cycles, the mean investigationtime was not significantly different between rats in pro-estrus (n = 9), in estrus (n = 8), in diestrus (n = 7) or afterovariectomy (n = 24), for the first (ANOVA, F(3,44) = 1.77,P = 0.160) and second (ANOVA, F(3,44) = 1.95, P = 0.13)encounters. Also, when we analyzed ELF EMF-stimulatedrats at 30-min IEI, in two phases of the estrous cycle, themean investigation times of the rats in pro-estrus (n = 4)were not significantly different from those in diestrus(n = 4) neither in the first encounter (P = 0.72) nor in thesecond encounter (P = 0.98). Although a role for estradiol isevident in the modulation of the social recognitionbehavior in ELF EMF-stimulated adult female rats (Fig. 2),we observed no differences in the mean investigationtimes between different estrous cycle phases or followingovariectomy, consistent with the report by Engelmann etal. (1998).

The effects of ELF EMF and ovariectomy on spontaneousbehavior and locomotor activity were also determined in the

Fig. 3 – Mean investigation times from OVX rats (left panel)and OVX plus E2 replacement (right panel). The amount ofinvestigation directed to the juvenile during the secondencounter was not significantly different during the first one,in both conditions.

Fig. 2 – (A) The effects of ELF EMF-stimulation on socialrecognition responses in ovary-intact adult female rats. (B)The effects of ELF EMF-stimulation on social recognitionresponses on OVX female rats without and with E2. 300-minafter the first encounter (solid bars) the adult rats wereexposed to the same juvenile during the second encounter(grey bars). Both ELF EMF and OVX ELF EMF + E2 groupsshowed a significant reduction of the mean investigationtime during the second encounter, t test, P = 0.002 (A, B, rightpanels). In contrast, non-ELF EMF-stimulated and OVX ELFEMF-stimulated groups did not show significant differencesbetween first and second encounters (A, B, left panels).

Table 2 – Description of experimental groups

IEI(min)

Non-ELF EMF-stimulated groups

ELF EMF-stimulatedgroups

Intact OVX OVX + E2 Intact OVX OVX + E2

30 1a 1b 1c 1d300 2a 2b 2c 2d 2e 2f

Non-ELF EMF stimulated groups and ELF EMF-stimulated groups.Each group was divided into 5 subgroups (n = 8), and they were testedin the social discrimination procedure with an inter-exposureinterval (IEI) of 30 or 300 min.

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ten different experimental groups (Table 3). We did not finddifferences in the median of the total activity counts amongthe 10 groups, which ranged between 40 and 67.5, P = 0.60(Kruskal–Wallis test). Therefore, we concluded that neitherELF EMF irradiation nor ovariectomy affected locomotoractivity.

3. Discussion

The present study provides the first demonstration of aninteraction between estradiol and ELF EMF in improving socialrecognition behavior in female adult rats. Our results showthat in both gonadally intact females and OVX females treatedwith E2, ELF EMF treatment prolongs the duration of a short-term social recognition memory which remains intact for upto 300 min following exposure to a stimulus juvenile. This didnot occur in OVX animals that were not treated with E2. Theseresults show that females as well as males (Vázquez-García etal., 2004) show enhanced retention of this form of socialrecognition memory following ELF EMF although hormonedependency has not yet been investigated in males.

In this paper, the results obtained for the non-ELF EMF-stimulated group (Fig. 1A) show that the social recognitionbehavior had similar properties to those reported previously(Ferguson et al., 2002; Thor and Holloway, 1982). In

Table 3 – Locomotor activity during first and secondencounters for all groups tested

Groups IEI(min)

n Firstexposure

Secondexposure

Non-ELF EMF 30 8 62.5 48.0ELF EMF 30 8 52.0 60.0OVX non-ELF EMF 30 8 47.5 42.5OVX-ELF EMF 30 8 65.5 67.5Non-ELF EMF 300 8 65.0 40.0ELF EMF 300 8 62.0 58.5OVX-ELF EMF 300 8 48.0 51.5OVX-ELF EMF + E2 300 8 57.0 44.5OVX non-ELF EMF 300 8 64.5 59.5OVX non-ELF EMF + E2 300 8 62.0 53.0

Values are median of total activity counts. These results indicatethat rats did not differ in activity within any experimental group,P = 0.603 (Mann–Whitney U test).

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agreement with those reports, we found a significantdecrement in the time spent by adult female rats investigat-ing juvenile female rats when re-exposed to them after an IEIof 30 min but not after 300 min (Ferguson et al., 2002; Thorand Holloway, 1982). While the gonadally intact rats receiv-ing ELF EMF showed an unexpected overall decline ininvestigation times at the 30-min test, this did not occur inOVX or OVX + E2 treated animals, and it seems unlikelytherefore that ELF EMF treatment has any major significanteffects on investigation times. Certainly there was no effectmaintained at the 300-min IEI.

In the current experiments, we were unable to find anyclear evidence for hormonal influences on social recognitionmemory performance either in the context of the estrus cycleor following ovariectomy or E2 treatment. Nevertheless, thefacilitatory effects of ELF EMF were estrogen-dependent.Other groups have also failed to find significant hormonalinfluences in rats on this task (Engelmann et al., 1998).However, Tang et al. (2005) have demonstrated that estrogenreplacement in ovariectomized mice can preserve socialrecognition for at least 24 h. It is possible that increasingthe difficulty of the task by reducing initial exposure times ortesting with IEIs between 30 min and 300 min might revealsome hormonally mediated facilitation. However, socialrecognition ability is of constant relevance for identifyingothers as familiar or unfamiliar, to trigger aggression towardsunfamiliar intruders, to assess social and reproductive statusand thereby promote species survival (Choleris et al., 2004;Ferguson et al., 2002; Thor and Holloway, 1982; Winslow andInsel, 2004). These functions are clearly important at all timesduring an animal's life and one might not therefore expectthat natural hormonal changes would have major effects onrecognition abilities. However, the fact that ELF EMF onlyproduced effects when estrogen is present, either in intactanimals or in ovariectomized animals treated with estradiol,does suggest that this hormone must have some facilitatoryrole. It would therefore be important to continue to investi-gate the possibility of direct facilitatory effects of estrogens,or other sex hormones, on social recognition memory. In thisrespect, it is interesting that female rats do seem to maintaina longer recognition memory (up to 120–180 min) than males(Dluzen et al., 2000; Engelmann et al., 1998; Gheusi et al.,1994).

The neuropeptides, oxytocin and vasopressin, are knownto have a role in social recognition in both female and malerats and mice (Choleris et al., 2004; Dantzer et al., 1988;Dluzen et al., 1998; Engelmann et al., 1998; Ferguson et al.,2002; Sekiguchi et al., 1991; Young, 2002). There is evidenceindicating that estrogens play an important role in regulatingthe action of oxytocin on short-term social recognition infemale rats and mice (Choleris et al., 2004; Dellovade et al.,1999; Quiñones-Jenab et al., 1997). It is therefore possible thatELF EMF is in someway facilitating their release in responseto social cues in the presence of estrogen (Choleris et al.,2003; Hlinak, 1993; Imwalle et al., 2002). However, at thisstage, we do not know what specific mechanism leads tocombined ELF EMF and estrogen effects, and there are clearlymany possible estrogenic actions via changes in neuralexcitability as well as through genomic actions (Wong andMoss, 1992). Further studies are needed to investigate this.

Our present results cannot establish directly whether ELFEMF, or the interaction between ELF EMF and E2, is due toeffects at the acquisition or retrieval levels. However, themostlikely explanation would seem to be improved consolidationof the memory post-acquisition although it will be importantto investigate whether just applying ELF EMF prior to a recalltest can also improve performance.

So far as we can judge, spontaneous behavior andlocomotion in female Wistar rats is not altered either by ELFEMF stimulation (Pesic et al., 2004; Vázquez-García et al., 2004)or by ovariectomy (Sato et al., 2003) nor by estrus cycle (Perrottiet al., 2001).

In conclusion, although our present results do not allow usto identify the mechanisms by which estrogens and ELF EMFlengthened social recognition memory, since estrogen recep-tors are present at high density in the olfactory bulb andamygdala (Guo et al., 2001; Kuiper et al., 1997; Shughrue et al.,1998), we suspect that they play an important role in theimprovement in the social recognition performance of femalerats produced by ELF EMF stimulation.

4. Experimental procedures

4.1. Animals

A total of 160 naive female Wistar rats (80 adults and 80juveniles) from our colony were used. The adults weighed262.67 ± 33.64 g and the juveniles weighed 118.06 ± 9.20 g. Priorto surgery, adult ratswere group housed (5–6 animals per cage)with free access to food and water and maintained on anartificial light-dark cycle (12:12 h, lights on at 0800 h). Thejuvenile females, 20–22 days old, were used as social stimuli.Adult and juvenile animalswere caged individually 1 h prior tothe social recognition tests and during the inter-exposureinterval (IEI). In adult male rats, stimulation with ELF-EMFincreases the duration of memory to 300 min after originalexposure to a juvenile conspecific (Vázquez-García et al.,2004). Therefore, in the present study, adult female rats weretested with two different IEIs, 30 or 300 min. All theexperiments were conducted during the light phase of thecycle, between 0900 and 1500 h.

4.2. Experimental groups

Group 1. These animals (n = 32, 16 intact and 16 ovariecto-mized) were submitted to an IEI of 30 min. The group wasdivided into 4 subgroups: (1a) intact and non-ELF EMF-stimulated (non-ELF EMF-stimulated), (1b) ovariectomizednon-ELF EMF (OVX-non ELF EMF), (1c) intact and ELF EMF-stimulated (ELF EMF-stimulated), and (1d) OVX ELF EMF-stimulated (Table 2).

Group 2. These animals (n = 48, 16 intact and 32ovariectomized) were tested with an IEI of 300 min. Thegroup was divided into 6 subgroups: (2a) intact and non-ELFEMF (non-ELF EMF), (2b) OVX-non-ELF EMF, (2c) OVX-non-ELFEMF and injected with E2 (OVX-non-ELF EMF + E2), (2d) intactand ELF EMF-stimulated (ELF EMF-stimulated), (2e) OVX-ELFEMF-stimulated, and (2f) OVX-ELF EMF-stimulated andinjected with E2 (OVX-ELF EMF + E2) (Table 2).

Fig. 4 – Schematic representation of ELF EMF-stimulation.A is ELF EMF stimulation or non-ELF EMF stimulation by9 days; B is first training day; C is second training day,and D is testing day.

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4.3. Surgery

Forty-eight adult rats were anesthetized with chloral hydrate400 mg/kg IP and subsequently ovariectomized using adorsolateral approach under aseptic conditions. After surgery,theywere housed in standard plastic cages in pairs for 15 days,and then they were group-housed (5 animals) with theirnormal cage mates in the exposure chamber. Vaginal smearswere taken daily for 20 days after surgery. Three days beforethe social recognition test, and 22 days after surgery 16 ratsreceived a daily subcutaneous injection of 17-β-estradiol(Sigma), 10 μg/day, for 3 days. The injections were givenprior to training and testing sessions. The dose of estradiolchosen produces circulating estradiol levels within thephysiological range and induces behavioral estrus in OVXfemales (Leuner et al., 2004).

All efforts were made to minimize the number of animalsused and their suffering. All procedures were performed instrict accordance with the guidelines and requirements of theEthical Committee of the National Autonomous University ofMexico.

4.4. ELF EMF exposure

Electromagnetic fields were generated inside the exposurechamber with a pair of circular Helmholtz coils as previouslydescribed (Vázquez-García et al., 2004). In brief, the two coilswere connected in parallel to minimize the total impedance ofthe wire and to map the magnetic field. They were connectedto a 120 V adjustable transformer (Staco Energy Products, Co.Dayton, OH, U.S.A). The sinusoidal magnetic flux density of1 mT was measured using a hand-held Gauss/Tesla Meter(AlphaLab, Inc). Helmholtz coils provide a very uniform fieldover a relatively large volume in the space between the coils(Kirschvink, 1992; Trout, 1988).

Coils were spaced apart at a distance equal to their radii inthe upper and lower face of the plastic exposure chamber(30 × 30 × 15 × cm). ELF EMFwere applied daily for 2 h from 0900to 1100 h for 9 days. This regime of stimulation was chosenbecause it induces cellular differentiation (Drucker-Colin et al.,1999) and was the one we have previous shown to increaseretention of social recognition memory in male rats (Vázquez-García et al., 2004). The exposure chamber housed 5 ratsduring each experimental session. Non-ELF EMF-stimulatedanimals were maintained for an equal period of time insidethe exposure chamber with the coils turned off. The magneticfield ambient background level was <0.04 mT.

Experiments were carried out at average room temperature(25 ± 2.0 °C). The temperature inside the exposure chamberwas 25.4 ± 0.4 °C and calculated illumination intensity was 350lux.

4.5. Social recognition

The social recognition procedure used was similar to thatdescribed previously (Thor and Holloway, 1982; Vázquez-García et al., 2004). The program of stimulation and testingwas as follows: starting on day 7 of ELF EMF stimulation ornon-ELF EMF stimulation, adult rats were trained for 2 daysand tested on day 9 (Fig. 4).

At each training session, individual adult rats wereintroduced into the test cage (50 × 50 × 42 cm) for a habituationperiod of 4 min. Each testing session consisted of a sequenceof four 4-min trials. The first trial was the first habituationperiod of the adult rat; the second trial was the first encounterbetween the adult rat and the juvenile rat, i.e., the firstinteraction in the social recognition test; the third trial was thesecond habituation period; the fourth trial was the re-exposure to the same juvenile stimulus after an inter-exposure interval (IEI) of either 30 min or 300 min. Followingeach encounter trial, the test cage was thoroughly cleaned. Inevery session, a subject was tested twice with the samejuvenile stimulus animal, and none of the animals were usedmore than once.

The effects of estrous cycle on social recognition wereinvestigated in adult rats that remembered a previouslyexposed juvenile (56 out of 80 adult female rats from non-ELF EMF and ELF EMF animals described above (Table 2)) Basedon vaginal smear cytology, intact animals were divided intopro-estrus (n = 9), estrus (n = 8) and diestrus (n = 7). Also, OVXrats (n = 24) were included in this analysis.

Video recording of investigatory behavior was used toassess the time spent by adult rats investigating thestimulus animal in the social recognition test. The datacollected from video-recordings were transferred to an IBM-PC computer for off-line analysis. Behaviors considered to berelated to social recognition learning and memory wereanogenital sniffing, close following, and pawing of thestimulus animal (Thor and Holloway, 1982). The differencein the mean durations of these behaviors between the twoencounters was taken as an index of social recognitionmemory. In addition, we computed the ratio index bydividing the mean investigation times of the secondencounter by those of the first one (Ferguson et al., 2001;Hlinak, 1993).

4.6. Locomotor activity

Locomotor activity was measured as previously described(Hotsenpiller and Wolf, 2003; Vázquez-García et al., 2004) byrecording the number of line crossings in an open-fieldapparatus. The open-field apparatus was the 50 × 50-cm testcage with a white floor divided into nine squares(16.6 × 16.6 cm each). Locomotor activity reported wasmeasured during the first and the second encounter on the

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test day for each rat. The summation of all line crossings istermed the total activity count.

4.7. Statistical analysis

Throughout, pooled data are presented as mean ± SEM. All Pvalues were calculated from paired t tests; P < 0.05 wasconsidered as significant. Effects of ELF EMF on socialrecognition were assessed with one-way analysis of variance(ANOVA), and Mann–Whitney U tests were used to analyzelocomotor activity. Post hoc comparisons were conductedusing Dunnett's test and Dunn's test.

Acknowledgments

We are indebted to Dr. Robyn Hudson for critically reading themanuscript, and Mr. Humberto Zarate-Alvarado, Mr. RobertoReyes-Cruz and Mr. Fernando Carvajal-Esquivel for theirexcellent technical assistance. Supported by DGAPA-UNAM,Grants IN-200502, IX231304 and IN-202302.

R E F E R E N C E S

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