Maternal Hyperthyroidism in Rats ImpairsStress Coping of Adult Offspring

Limei Zhang,1* Vito S. Hernandez,1 Mauricio Medina-Pizarro,1

Pablo Valle-Leija,1 Arturo Vega-Gonzalez,1 and Teresa Morales2

1Departamento de Fisiologıa, Facultad de Medicina, Universidad Nacional Autonoma de Mexico,Mexico DF, Mexico2Departamento de Neurobiologıa Celular y Molecular, Instituto de Neurobiologıa,Universidad Nacional Autonoma de Mexico, Queretaro, Mexico

Given the evidence that maternal hyperthyroidism (MH)compromises expression of neuronal cytoskeletal pro-teins in the late fetal brain by accelerated neuronal dif-ferentiation, we investigated possible consequences ofMH for the emotional and cognitive functions of adultoffspring during acute and subchronic stress coping.Experimental groups consisted of male rat offspringfrom mothers implanted with osmotic minipumps infus-ing either thyroxine (MH) or vehicle (Ctrl) during preg-nancy. Body weight and T4 level were monitored duringthe first 3 postnatal months, and no differences werefound with the controls. We analyzed hippocampal CA3pyramidal neurons and dentate granular cell morphol-ogy during several postnatal stages and foundincreased dendritic arborization. On postnatal day 90 amodified subchronic mild stress (SCMS) protocol wasapplied to experimental subjects for 10 days. The Mor-ris water maze was used before, during, and afterapplication of the SCMS protocol to measure spatiallearning. The tail suspension test (TST) and forced-swimming test (FST) were used to evaluate behavioraldespair. The MH rats displayed normal locomotor activ-ity and spatial memory prior to SCMS, but impairedspatial learning after acute and chronic stress. In boththe FST and TST we found that MH rats spent signifi-cantly more time immobile than did controls. Serumcorticosterone level was found to increase after 30 minof restraint stress, and corticotropin-releasing factorimmunoreactivity was found to be increased in the cen-tral nucleus of the amygdala. Our results suggest thatMH in rats leads to the offspring being more vulnerableto stress in adulthood. VVC 2007 Wiley-Liss, Inc.

Key words: depression; spatial learning; corticotropinreleasing factor; corticosterone

Hormones markedly affect neuronal structure andfunction in various ways throughout life. During preg-nancy, maternal thyroid hormones [triiodothyronine(T3) and thyroxine (T4)] cross the placenta in rat(Obregon et al., 1984; Morreale de Escobar et al., 1988)and human (Vulsma et al., 1989) and are postulated toregulate fetal brain development. The relatively inactive

T4 is converted to the more active form, T3, in braintissue by the action of the enzyme deiodinase type II(Tanaka et al., 1981; Courtin et al., 1988). Thyroid hor-mone (TH) appears to regulate those processes associatedwith terminal brain differentiation such as dendriticand axonal growth, synaptogenesis, neural migration,and myelination (for a review, see Oppenheimer andSchwartz, 1997). TH also plays a significant role in theproliferation and survival of brain cells. A deficiency ofTH alters the expression of several members of the Bcl-2 family, down-regulating proapoptotic genes and up-regulating antiapoptotic ones (Singh et al., 2003), andincreases pro–nerve growth factor and p75 neurotrophinreceptor levels associated with enhanced apoptosis(Kumar et al., 2006) in developing rat cerebellum andcerebral cortex, respectively.

Most studies of prenatal thyroid disorders, both inhumans and in rats, have centered on hypofunctionbecause of the high incidence of hypothyroidism and thesevere consequences it has for offspring. Nevertheless,maternal hyperthyroidism (MH) is a significant endo-crinologic disorder in pregnancy. The prevalence of thy-rotoxicosis, predominantly Graves’ disease, has beenreported to be 0.05–0.2%, with an additional 3% ofmothers exhibiting gestational transient thyrotoxicosis(Burrow, 1993; Glinoer, 1997; Polak et al., 2006).However, according to clinical reports, only 1% of chil-dren born to women with Graves’ disease are describedas having hyperthyroidism, and overt neonatal hyper-thyroidism is rare and concerns only 1 of 50,000 neo-nates (Polak, 1998). On the other hand, studies in ananimal model of MH have shown that this condition

Contract grant sponsor: CONACYT; Contract grant number: 46141-M(to L.Z.); Contract grant sponsor: DGAPA-UNAM; Contract grant num-bers: IN210406 (to L.Z.), IN224407 (to A.V.G.), IN205706 (to T.M.).

*Correspondence to: Limei Zhang, MD/PhD, Department of Physio-logy, Faculty of Medicine, UNAM, Mexico City 04510, Mexico D. F.E-mail: limei@servidor.unam.mx

Received 3 May 2007; Revised 4 July 2007 and 24 August 2007;Accepted 16 September 2007

Published online 11 December 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jnr.21580

Journal of Neuroscience Research 86:1306–1315 (2008)

' 2007 Wiley-Liss, Inc.

compromises expression of neuronal and astrocyticcytoskeletal proteins in the late fetal brain, suggestingaccelerated neuronal differentiation (Evans et al., 2002).This aberrant timing of the central nervous system(CNS) development might lead to subtle but irreversiblechanges in its synaptic wiring, which could exert crucialinfluences on neural plasticity in adulthood, although theindividual might be seen as normal in an unaltered situa-tion. Despite numerous studies on the molecular andneurochemical effects of hyperthyroidism in the prenatalperiod, little is known about the long-term consequen-ces of such postulated early-developmental accelerationof the CNS in the adulthood of offspring.

Any physical or psychological stressor that threatensthe homeostasis of an organism can initiate a set ofbehavioral and neuroendocrine responses intended to helpthe organism to adapt to the altered situation. In conduct-ing this neuroendocrine response, the limbic-hypothal-amo-pituitary-adrenal axis and the sympathoadrenal axisare two major pathways mediating the major compo-nents of the stress response (Gulpinar and Yegen, 2004).Recent opinions on stress emphasize that there are indi-vidual differences and several response patterns in copingwith these challenges (McEwen and Sapolsky, 1995).The role of stress in induction, maintenance, and relapseof psychiatric dysfunction is well established, and there isgood evidence that changes in glutamatergic and dopa-minergic neurotransmission in the prefrontal cortex andhippocampus may be responsible for behavioral abnor-malities seen in emotional disturbances (Moghaddam,2002). A recent hypothesis on the pathophysiology ofdepressive disorders involves adaptive plasticity of theneural system. As proposed by Duman et al. (1999),depression could result from an inability to make theappropriate responses to stress, as a consequence of adysfunction of the normal mechanisms underlying neuralplasticity.

The goal of the present study was to investigatewhether MH produces anatomocytochemical relativelystable lifelong changes during development that couldinfluence or alter the adulthood behaviors of offspring,especially their vulnerability to stress. We used the well-validated chronic mild stress protocol to produce mildand unpredictable daily stressful situations for the rats.This protocol is regarded as closely modeling the humansituation, consisting more of daily hassles than traumaticevents (for reviews, see Willner, 1997, 2005).

MATERIALS AND METHODS

Animals

Wistar rats from the animal house of our Faculty ofMedicine were used in this study. All animal procedures wereperformed in accordance with the principles presented in the‘‘Guidelines for the Care and Use of Mammals in Neuro-science and Behavioral Research’’ by the National ResearchCouncil. Ten postnatal day 90 (P90) female rats wereimplanted subcutaneously with Alzet osmotic pumps (Model2ML4, pumping rate 2.5 lL/hr for 28 days) infusing either

thyroxine (to progenitors of MH subjects) or vehicle [to pro-genitors of control (Ctrl) subjects]. The dose of thyroxine(T4; T2501, Sigma-Aldrich Inc.) was 1.5 lg/100 g of premat-ing body weight per day (for detailed chemical preparationprocedures, see Evans et al., 2002). With this dose, the femalerat serum T4 concentration was about 2.26 6 0.14 ng/dL(n 5 4) , whereas that of the control females was about 1.346 0.11 ng/dL (n 5 4). This measurement was made with thechemiluminescent microparticle immunoassay (CMIA,ARCHITECT1, Abbott Laboratories, Abbott Park, IL). Aftera 2-day recovery period, females were mated with normalmales. Osmotic pumps were removed from the females onoffspring postnatal day 1 (P1). At the end of lactation (off-spring P30), four male offspring rats per progenitor were ran-domly chosen to form either the MH group (n 5 20) or theCtrl group (n 5 20). Further selection for experimentalmanipulations was performed randomly. All animals weremaintained in an artificial 12-hr light:12-hr dark cycle (lightson at 16:00) in a room at 228C with food and water ad libi-tum, except when the subchronic mild stress protocolrequired deprivation. Body weight was monitored, and plasmaT4 level was measured with the CMIA microparticle immu-noassay as above mentioned.

Experimental Design and Stress Procedure

Experimental procedures were performed when ratswere in the young adult stage (starting at P90, body weight350 6 10 g) except for morphological analysis, in which brainsamples were collected at P7, P30, and P75 and processedwith the Golgi-Cox impregnation method (see detaileddescription below). A modified subchronic mild stress proto-col (SCMS) lasting 10 days was used in this study (Table I).The regimen consisted of the application of a variety ofunpredictable stressors, one per day, which included: 30 minof restraint stress (confinement into a cylinder 20 cm long and7.5 cm in diameter), habitat changing with increased light andnoise, one light cycle of sleep deprivation (four rats housed incages 39 3 75 3 34 cm with a 3-cm water level and fourislands made of cylinders 8 cm in height and 7.5 cm in diame-ter), 1 day of food and water deprivation, one 10-min periodof electric foot-shock stressor (0.2 mA, 0.5-sec pulse, 2 permin over 10 min for a total of 20 shocks; Heinrichs andKoob, 2005). The behavioral and cognitive tests specifiedbelow also served as unpredictable stressors during the 10-dayprotocol. A summary of the experimental design is shown inTable I.

Morris Water Maze

The Morris water maze (MWM; Morris, 1984) assessesspatial learning and memory retention. A black circular pool(diameter 156 cm, height 80 cm) filled with 30 cm of water(258C 6 18C) with visual cues was used for this cognitivetest. A circular black escape platform (diameter 12 cm) wassubmerged 1 cm below the water surface. Rats were allowedup to 120 sec to locate the escape platform. If the allowedtime was finished and the experimental subjects had not foundthe platform, they were guided to the platform. Once on theplatform, rats were permitted to stay for 5 sec to allow them

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to observe place and location. To assess cognitive function instress-naive experimental subjects and any possible differencein response to this mild swimming stress, rats were not pre-trained for the first cognitive test (nonstress situation) on day1 of the SCMS protocol. The second MWM test was per-formed on day 3 of the SCMS protocol, when the rats hadundergone an immobilization stressor on day 2 and habitatchanging stress with increased light and noise for 2 hr prior tothe cognitive test during the endogenous activity period. Thiswas defined as an acute stress situation. A third MWM testwas performed on day 10 of the SCMS protocol and desig-nated as a subchronic stress situation. The memory retentiontest of the MWM was performed on day 11, when the plat-form was removed, and the time spent on the platform quad-rant was recorded. The starting position was in the quadrantopposite (IV) the platform quadrant (II) and was unchangedfor the four tests.

Repeated Forced-Swimming Stress

Animals from the MH and Ctrl groups were assessed fordepression-like behavior using a modified version of theforced-swimming test (FST; Porsolt et al., 1978; Wellmanet al., 2007), in which experimental subjects were exposedrepeatedly to the swimming stressor during the dark period ofthe artificial light–dark cycle. The repeated FST allowed us toevaluate depression-like behavior after both acute and repeatedexposure to the test while acting as a stressor and helped torule out the possibility of any motor impairment. The testconsisted of exposure to a vertical Plexiglas cylinder 45 cm inheight and 30 cm in diameter containing water to a height of25 cm kept at 258C 6 18C. On P60, the rats underwent thefirst FST for 6 min to assess this behavioral parameter prior tothe SCMS protocol. We designated this as the baseline test.On P96, day 1 of the test, rats received a single 15-min FST.On day 2 of the test, the rats received four consecutive 6-minexposures with a 12- to 15-min interval between trials. Passiveimmobility (cessation of spatial displacement with or withoutminor involuntary movements of the hind limbs) during min

2–6 was analyzed off-line. The observer scored the rats as ei-ther ‘‘swimming’’ or ‘‘immobile’’ every 5 sec (sampling fre-quency criterion described by Detke et al., 1995), and thepercentage of immobility episodes was calculated as immobil-ity episodes observed/total number of observations 3 100.Immobility counts in the FST are regarded as a measure ofbehavioral despair.

Tail Suspension Test

In the tail suspension test (TST; Chermat et al., 1986),rats are suspended by the tail for 6 min during which theyshow periods of agitation and immobility. Duration of immo-bility is measured as an indicator of behavioral despair as anadditional test to confirm the depression-like behavior dis-played in the FST. The tail suspension apparatus consisted ofa wooden cubicle with inside dimensions of 25 3 25 3 55cm. A cylinder (5 cm in diameter, 25 cm in length) was fixedinside the cubicle (5 cm from the top) as a tail hanger. Ratswere suspended from the cylinder using adhesive tape placedat about half the total tail length. Compared with the tradi-tional design, in which a hook is used as a tail hanger, theTST apparatus used in this study avoided possible harm to therats’ tails. The entire test was video-recorded, and total immo-bility time was analyzed off-line.

Corticosterone Measurement

Pre- and postrestraint stress (30 min) serum corticoster-one (CORT) level during the rats’ late activity period wasdetermined. Rats from the MH and Ctrl groups were anesthe-tized with an overdose of sodium pentobarbital and cardiacblood samples were collected and allowed to coagulate atroom temperature for 3 hr. Samples were subsequently centri-fuged at 2,000g for 15 min. Serum was removed and stored at2208C until analysis. Serum CORT concentration was deter-mined by a commercially available ELISA kit according to themanufacturer’s instructions (Assay Designs Inc., Ann Arbor,

TABLE I. General experimental design

Postnatal Stage Experimental day Experimental procedures Parameters measured

P60 Forced swimming test (n 5 9). Immobility counts (Baseline)P90 lst of SCMS Morris water maze (MWM) (n 5 9). Escape latencyP91 2nd Restraint stress (30 min) (n 5 14), followed by

cardiac blood sample collection (n 5 5)a.Serum Corticosterone, T4 levels.

P92 3rd Habitat changing with increased light and noise for2 hrs prior to MWM. MWM (n 5 9 for allremaining procedures).

Escape latency.

P93 4th Sleep deprivation during light cycle.P94 5th Food and water deprivation for 24 hrs.P95 6th Electric footshock stressor.P96 7th Repeated forced swimming stress (RFSS) day 1, for

15 min.Immobility counts

P97 8th RFSS day 2, 6 min 3 4 trials. Time between trials:12–15 min

Immobility counts

P98 9th Tail suspension test (TST) Immobility timeP99 10th MWM Escape latency.P100 11th MWM Perfusion for immunocytochemistry Swimming time on platform area

aMWM at day 1 was not applied to these animals.

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MI). Serum dilution was 1:10. Each sample and standard wasmeasured in duplicate.

Histological Procedures for Corticotropin-ReleasingFactor Immunoreaction

After the MWM memory retention test, rats weretreated with an overdose of sodium pentobarbital and perfusedtranscardially with 0.9% saline followed by cold fixative con-taining 4% paraformaldehyde in 0.1M sodium phosphatebuffer (PB; pH 7.4) plus 15% (v/v) saturated picric acid for 15min. Brains were removed, blocked, and then thoroughlyrinsed with PB. Coronal sections (50 lm) of amygdala andhypothalamus were obtained using a vibratome (Leica VT1000, Heidelberg, Germany). Free-floating sections werequenched by incubating brain sections in 3% hydrogen perox-ide in PB for 10 min. All the sections for immunoreactionwere then incubated with PBST: PB 1 0.9% NaCl 1 0.3%Triton X-100 (T-7878, Sigma-Aldrich, Inc.) and 20% normalgoat serum (NGS; 005-000-121, Jackson ImmunoResearchLaboratories, Inc.) for 1 hr at room temperature (RT). Rabbitantiserum anti–corticotropin-releasing factor (CRF; AP1760,Chemicon International Inc., CA) was used as primary anti-body diluted 1:1,000 in PBST 1 1% NGS and incubatedovernight in a cold room. Afterwards, sections were rinsed for10 min three times with PBST and then incubated for 4 hr atRT with biotinylated goat antirabbit secondary antibodies(1:200; Vector Labs, Burlingame, CA). Finally, sections wereincubated in avidin-biotin-peroxidase complex (Elite ABCkit, Vector Labs) for 1 hr at RT. Peroxidase was detectedusing diaminobenzidine (DAB) as chromogen. Sections weredeveloped using a Liquid DAB-Plus Substrate Kit (00-2020;Zymed Laboratories, San Francisco, CA). For indirect immu-nofluorescence Alexa Fluor 488 goat antirabbit IgG was usedas a secondary antibody (A11008, Molecular Probes, Eugene,OR).

Image Analysis

The central nucleus of the amygdala (CeA) and paraven-tricular nuclei (PVN) were analyzed in regions spanningbregma 21.60 and 23.30 mm and 21.80 and 22.12 mm,respectively, according to the Paxinos and Watson brain atlas(Paxinos and Watson, 1998). Immunoreactive (IR) cell count-ing was performed using a Nikon Eclipse 50i microscope witha 403 objective lens. The IR-positive cells were projectedand marked into a defined area (being circles 50 lm in diame-ter for CeA and 100 lm in diameter for PVN) through adrawing tube (Nikon Y-IDT) at 38 magnification. Countingwas performed manually, and data were then normalized.

Golgi-Cox Impregnation

For the Golgi-Cox impregnation procedures, on P7,P30, and P75 two rats from each group received an overdoseof anesthesia and were decapitated. Brains were removed fromthe skulls quickly, and their central thirds (along the antero-posterior axis) were cut with a sharp blade into blocks approx-imately 5 mm thick. Tissues were briefly rinsed with 0.1MPB (pH 7.4) and then immersed in sequenced impregnationsolutions (FD Rapid GolgiStain kit, FD Neuro Technologies,

Ellicott City, MD) for 2 weeks in the dark. Sections (100 lm)were sliced using a vibratome and were dried naturally at RTin the dark and then stained with a solution provided withthe stain kit. Dendritic patterns of dentate granule cells andCA3 pyramidal neurons were reconstructed using a drawingtube at 3400 magnification. Dendritic arborization areas intwo dimensions were estimated using Adobe Photoshop(Adobe Systems Inc.) and Fovea Pro (an image tool kit fromReindeer Graphics, Asheville, NC).

Statistical Analyses

Quantitative results were expressed as mean 6 standarderror of mean (SEM). Groups were tested for differences byanalysis of variance followed by the Student-Newman-Keulstest, using InStat (GraphPad Software, San Diego, CA). Dif-ferences were considered statistically significant at P < 0.05(*P < 0.05, **P < 0.01, and ***P < 0.001, versus the con-trol group).

RESULTS

Spontaneous Locomotor Activity, Body Weight,and T4 Serum Level Are Unaffected in MH Rats

MH rats displayed normal locomotor activity dur-ing the manipulations and different tests. Their bodyweight was similar to that of the Ctrl rats and on P90was 350 6 10 g. The measured mean serum T4 concen-tration was 1.26 6 0.14 ng/dL, which was not signifi-cantly different from that of the control (1.17 6 0.06ng/dL).

Altered Serum CORT Level in MH Rats afterAcute Stress

To monitor the HPA axis response to an acuterestraint stress, serum CORT levels were assessed bycomparing MH animals with controls. First, we mea-sured serum CORT level in the rats’ late-activity periodbefore any stressor was applied. The mean serumCORT concentrations without restraint stress of thecontrol and MH groups—3.303 6 0.416 and 3.029 60.617 lg/dL (n 5 4), respectively—did not differ signif-icantly. Rats were subjected to acute restraint stress (30min) in their late activity period, when the intrinsic cir-cadian amplitude of CORT was expected to reach itsminimum (Fig. 1). The average CORT level of MHrats was 18.3 6 0.7 lg/dL, whereas that of the controlswas 9.4 6 3.0 lg/dL. This increase was consistent withpreviously reported levels in response to such a stressor(Viau and Sawchenko, 2002).

Increased Depressive-Like Behavior duringSCMS Application

The speed of development of behavioral despairunder the SCMS paradigm was assessed using two well-validated behavioral tests, the FST and the TST. Bothtests are based on rats having an immobility response toan inescapable adverse situation. Duration of immobility,reflected as frequency of immobility episodes of totalobservations in the FST, is understood to be a measure

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of behavioral despair with a direct relationship to it. Amonth before application of the SCMS protocol, ratswere subjected to the first FST for 6 min in order tohave a baseline reference for immobility (Fig. 2A, base-line). The MH rats had fewer immobility episodes thandid the rats from the Ctrl group. On day 7 of theSCMS protocol animals underwent 2 consecutive daysof the forced-swimming stress test (FSST; Wellmanet al., 2007). Rats from both experimental groups wereobserved to have a greater number of immobility epi-

sodes during this repeated forced-swimming test. Theimmobility count of the MH rats was much more pro-nounced (Fig. 2A, days 1 and 2). However, immobilitycounts of the MH and Ctrl rats did not differ signifi-cantly during the last exposure to the FST on the secondday. This lack of difference may suggest that both thecontrol and MH rats were approaching their physicaleffort limits. On the ninth day of the SCMS, the ratswere exposed to the TST for 6 min (Fig. 2B). Again,the mean duration of immobility of the MH animals waslonger (205.2 6 13.79 sec) than that of the Ctrl rats(128.6 6 37.10 sec).

Impaired Rat Spatial Learning and MemoryRetention during SCMS Application

The effects of the SCMS on spatial learning andmemory retention of the MH rats were tested using thehidden-platform water maze (Morris water maze,MWM). As shown in Figure 3A–C, the latency toescape to the platform of the Ctrl group decreased whileadvancing the trials. The slopes of the time sequences ofthe first three trials were relatively steep, approachingthe shortest latency at the third trial, regardless of theduration of application of the SCMS protocol. In con-trast, MH rats displayed progressively impaired spatiallearning while continuing the SCMS protocol. Figure3A shows the performance of naive MH rats in stressand swimming. The time sequence of the first four trialsrevealed a steep slope matching that of the control, sug-gesting normal spatial learning ability. However, theescape latency in the fifth and sixth trials peaked in thesequence and decreased again in the seventh and eighthtrials. This may be related to an overreaction of the

Fig. 2. Repeated forced-swimming stress test (FSST, A) and tail sus-pension test (TST, B) used to assess depressive-like behaviors. A:Baseline test (6 min) was done at P60 (n 5 9); the 2-consecutive-dayforced-swimming stress test (day 1 and day 2 tests) was done on days7 and 8 of the SCMS protocol with a duration of 15 min for the firstday, and 6 min for each trial on the second day with 12–15 min

between each trial (n 5 9). The percentage of episodes of immobilityof the total number of episodes observed is shown. In all cases, onlythe swimming behavior from the second to the sixth minutes was an-alyzed off-line. B: Total immobile time during the 6-min TST wasrecorded (n 5 9). Mean 6 SEM (***P < 0.001, **P < 0.01, *P <0.05).

Fig. 1. Serum corticosterone (CORT) levels determined to assess themagnitude of the stress reaction. Serum CORT level increases duringstressful events and is mainly regulated by HPA axis. Samples weretaken during the rats’ late-dark period before (prestress) and after(poststress) restraint stress (for 30 min). Data are given as mean 6SEM for n 5 5 (*P < 0.05).

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HPA axis to the novelty of swimming and handling gen-erated in the first trial of MH rats, although this noveltydid not seem to be a significant stressor for Ctrl rats.Figure 3B shows the performance of rats in an acutemild stress situation, which included habitat changingand increased noise and light 2 hr prior to the MWM.The slope of the MH rats was much smoother than thatof the control rats, and clear differences between theMH and Ctrl groups were observed. After the 10 daysof the SCMS protocol, the MH rats showed a MWMtime sequence with a totally different form than that ofthe naive and acute stress MH groups, with several peaksand almost no reduction in latency at the end of the test(Fig. 3C). Latency reduction in the Ctrl group wasdelayed in comparison with that in the naive and acutestress Ctrl rats but reached a similar level after the thirdtrial. In the quadrant preference test for memory reten-tion (Fig. 3D), the MH rats showed no preference forthe quadrant where the platform was. During the experi-mental periods, no rats showed any apparent sign of dis-comfort or locomotor disability.

Increased Number of CRF-IR Neurons in CeA

Immunocytochemical detection for CRF revealedan increased number of CRF-IR cells in the CeA ofMH rats subjected to the SCMS scheme (Fig. 4A–C).Positive labeling for CRF was detected in neurons inthe dorsal and ventral subdivisions of the medial parvo-cellular part of the caudal PVN. Also, a few cells wereobserved in the dorsal and lateral subdivisions of themagnocellular part (PaLM; Fig. 4D–F). These locationscorrespond with the typical areas of the PVN in whichCRF neurons have been described (Swanson and Saw-chenko, 1983). We found no significant differences inthe number of CRF-IR cells in these hypothalamicregions (Fig. 4F), although the expression pattern in MHrats seemed to be more scattered toward lateral hypo-thalamic regions (Fig. 4E,F). It is worth noting that thisstaining was performed in tissue from animals withoutprevious treatment with colchicine, which is known toexert an axonal transport blocking effect and helps thedetection of CRF or any other peptide by accumulationof antigens in the cell body.

Fig. 3. Morris water maze (MWM) used to assess spatial memory.Tests were performed with rats for different SCMS periods. A:Stress-naive rats on the first day of SCMS protocol. B: Rats under-went acute stress. C: Rats underwent 10 days of SCMS. D: Quad-rant preference test for memory retention performed on the 11th day

of SCMS. The platform was in quadrant II. Inserts show paths takenby representative rats with quadrant number indicated. Means 6SEMs (n 5 9) of escape latency across 8 trials represented in timesequences (**P < 0.01, *P < 0.05).

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Increased Dendritic Receptive Field inHippocampal Projection Neurons duringDevelopment and Adulthood

To elucidate the possible mechanisms underlyingthe behavioral changes, hippocampal CA3 pyramidalneurons and dentate gyrus (DG) granule cell dendriticarborization were analyzed through reconstruction ofGolgi-Cox-impregnated neurons on postnatal days P7,P30, and P75. A general observation is that the dendriticreceptive fields (2-D projection of 3-D arborization)were larger in the MH rats in all three stages, as shownby representative morphology of these neurons in Figure5. Using Adobe Photoshop and FoveaPro, we estimatedthe 2-D dendritic receptive fields, finding that at P75the value of the MH rats of 92.73 6 3.56 lm2 was sig-nificantly different from that of the Ctrl rats of 48.9 62.66 lm2 in CA3 pyramidal neuron apical receptivefields. Regarding DG granule cell dendritic receptivefields, no significant differences were found. However,thicker dendrite shafts were observed in samples fromthe MH rats. The inserts in Figure 5-P7 illustrate exam-ples of primary and secondary dendrite ramifications.

DISCUSSION

It is well established that thyroid hormone (TH)action early in life plays a key role in determining thenormal timing of neural development. An abundantnumber of studies focusing on excessive or insufficientTH during early development have revealed that this en-docrine imbalance leads to abnormalities in brain struc-ture. For instance, neonatal transient elevation of TH,from postnatal day 1 to postnatal day 4 in rats causes hy-pertrophic development of CA3 pyramidal neurons inthe hippocampus and of astroglial cells in the hippocam-

pus and basal forebrain (Gould et al., 1990a, 1990b).Our data describing an increase in the dendrite receptivefields in hippocampal CA3 pyramidal neurons at threepostnatal ages, P7, P30, and P75, are analogous to those

Fig. 5. Reconstruction of representative hippocampal CA3 pyramidalneurons and dentate gyrus granule cells impregnated by the Golgi-Coxmethod on postnatal day 7 (P7), postnatal day 30 (P30), and postnatalday 75 (P75). Scale bar5 50 lm for main picture and 10 lm for inserts.

Fig. 4. CRF-IR in the central nucleus of the amygdala (CeA) andthe paraventricular nuclei (PVN). A: Representative immunofluores-cence photomicrograph of CeA from Ctrl rats. Scale bar 5 50 lm.B: Representative immunofluorescence photomicrograph of CeAfrom MH rats. Scale bar 5 50 lm. C: Examples of CRF-IR neurons

visualized with the peroxidase method. Scale bar 5 20 lm. D:CRF-IR in PVN from Ctrl rats. Scale bar 5 100 lm. E: CRF-IRin PVN from MH rats. Scale bar 5 100 lm. F: Insert magnificationof E. Scale bar 5 20 lm. G: Histogram showing the number ofCRF-IR cells in CeA and PVN (n 5 5).

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seen in the rodent neonatal hyperthyroidism model andextend existing observations.

The present data are the first to describe the emo-tional and cognitive consequences of mild maternalhyperthyroidism in adult rat offspring under subchronicmild stress. Earlier studies concerning neonatal hyper-thyroidism have suggested that thyroxin accelerates thematuration of the pituitary-adrenal response to electricshock (Schapiro and Norman, 1967) and the onset ofphysiological and behavioral bioenvironmental interac-tion in general (Schapiro, 1968). It has also beenreported that neonatal thyroxin stimulation of albino ratsdisrupts hippocampal LTP, and despite hypertrophiedhippocampal CA3 pyramidal neurons and elevated den-sity of cholinergic neurons in the basal forebrain, theyare actually less efficient in learning a spatial maze (Pav-lides et al., 1991). The present finding of cognitiveimpairment in the MH rats, shown by their greaternumber of mistakes and increased latency in the Morriswater maze, is compatible with previous observations inneonatal hyperthyroid rats. Moreover, our data make animportant addition to previous observations in severalareas. The relatively mild degree of maternal hyperthyr-oidism used in this experimental design apparently didnot produce alterations in the adulthood of the offspringunder normal conditions, as we reported in the results.However, when the subchronic mild stress scheme wasapplied, their spatial learning capacity was progressivelyimpaired along the protocol. Furthermore, the display ofdepression-like behaviors by the MH adult offspringwas accelerated, as results of both the forced-swimmingstress test (FSST) and the tail suspension test (TST)showed. It is worth mentioning that depression-likebehaviors have multiple dimensions and that analysis ofsuch behaviors in an animal model is always difficult tosort out. Normally, a battery of tests is recommended toassess such behaviors. The forced-swimming test (FST) isprobably the most widely and frequently used protocol.In the present experimental design, we used a modifiedversion of the Porsolt test (Porsolt et al., 1978), mainlybecause repeated exposure to the test in a short time is astressor for rats. Hence, the depression-like behaviorcould be more quickly observed. On the other hand,the FSST demands a major physical effort by the animalunder study. A motor-impaired rat could be easily dis-tinguished during this repeated swim test. Both the FSTand TST have immobility as a parameter to be meas-ured. However, the actual parameter being measured isthe response of the animal to the specific condition ofeach test, that is, the adoption of floating behavior inthe FST or of stationary behavior in the TST. It hasbeen suggested that the observed behaviors might beproduced by different biological substrates, and differenthypotheses have been proposed to explain the observedimmobility in FST and TST (Cryan et al., 2005). Tosummarize these observations, our data suggest thatmaternal hyperthyroidism, however moderate or withoutapparent alteration in the adult offspring under normal

conditions, could bring about severe consequencesbecause of the enhanced stress response.

A stressful event, either physical or psychological,activates neural circuits involved in emotionalresponses, cognition, and homeostatic regulation. Thehippocampal formation and the amygdala form the cen-tral axis of the limbic system and are highly responsiblefor cognitive and emotional responses to a stressful sit-uation. The hippocampal formation receives majorinput from the entorhinal cortex through the perforantpathway, where highly processed sensory informationfrom associational, perirhinal, and parahippocampal cor-tices as well as the prefrontal cortex is transmitted. Theproximal segments of the apical dendrites of CA3 py-ramidal neurons are covered with complex spines orexcrescences that receive mossy fiber input from gran-ule neurons of the dentate gyrus (Blackstad and Kjaer-heim, 1961). The dentate gyrus–CA3 pathway providesthe major excitatory afferent to the hippocampal regioinferior, and each CA3 neuron excites multiple pyrami-dal neurons (Ishizuka et al., 1990). Our finding of aug-mentation of CA3 pyramidal neuron dendrite receptivefields most likely indicates that these excitatory path-ways to the hippocampus could be strengthened at thebeginning of a stressful situation, which amplifies sen-sory inputs from the entorhinal cortex. Therefore, thestressors could have a more profound effect on hippo-campal function. Ultrastructural studies showed thatchronic stress alters the synaptic terminal structure inthe hippocampus, especially the mossy fiber synapsesboutons en passant from granule neurons to the proxi-mal segments of apical dendrites of CA3 pyramidalneurons (Magarinos et al., 1997), indicating a possiblereorganization driven by hyperactivation of this path-way. Chronic exposure to higher levels of CORT cancause irreversible hippocampal dysfunction and increasedepression-like behavior in rats in a dose-dependentmanner and disrupt normal HPA axis function (Johnsonet al., 2006). Moreover, clinical evidence has shownthat medically healthy patients with recurrent majordepression can show decreased hippocampal volumeand impaired cognition (Sheline et al., 1999). Usingthe subchronic mild stress paradigm, we observed fasterdevelopment of depressive-like behaviors as well asimpairment of cognitive functioning in MH rats undermildly stressful conditions.

The neurohormone CRF activates both hormonaland behavioral responses to a variety of stressors. Stressleads to CRF release from the hypothalamic PVN,resulting in increased plasma corticotrophin (ACTH) andadrenal steroid concentration. CRF-containing cells con-stitute a significant neuronal population in the centralnucleus of the amygdala (CeA). The CeA has beenshown to be a key regulator of the stress response medi-ated by CRF release from the PVN (for a review, seeGray and Bingaman 1996). Discrete lesions of the CeAexacerbate the acute stress response (Carter et al., 2004).Our observation of an increased population of CRF-IRcells in the CeA but not in the PVN after being sub-

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jected to chronic mild stress might indicate a strongerattempt to limit the stress overreaction in MH animals.

The observations from this study raise the questionof the underlying mechanism by which mild maternalhyperthyroidism produces enhanced vulnerability to stressin adulthood. According to the current hypothesis of neu-ral plasticity in some areas of the neural system, once neu-ronal connections are established during this critical periodof brain development, the networks tend to be relativelystable (‘‘rigid’’ synaptic connections), whereas other typesof synaptic connections (‘‘flexible’’ synaptic connections)undergo lifelong self-optimization processes, so-called use-dependent neural plasticity. The cognitive, behavioral,and emotional reactivity of an individual derived fromthis kind of connection is stepwise remodeled to meetenvironmental demands. Although the presence of rigidsynaptic connections ensures stability of the principalcharacteristics of function, variable configuration of flexi-ble synaptic connections determines the unique, nonrep-eatable character of an experienced mental act (Arendt,2001; Gulpinar and Yegen, 2004). There are many possi-ble causes of aberrant synaptic connections. One possiblecause is hyper- or hypotrophied neuronal dendritic arbo-rization. In addition, neuronal migration aberrations can,in fact, alter physiological synaptic connections, resultingin loss of neural plasticity and consequently of adaptivecapacity. Auso et al. (2004) recently reported that a mod-erate and transient deficiency in maternal thyroid functionat the beginning of fetal neocorticogenesis alters neuronalmigration. The opposite could also alter neuronal migra-tion, although experimental data are required to confirmthis hypothesis.

In conclusion, the results from this animal modelanalyzing the consequences of maternal hyperthyroidismin the adulthood of offspring suggest that this delicateprenatal endocrine modification could alter the neuronalstructure of specific brain areas and influence the cogni-tion and emotionality of offspring in adulthood. Thesemodifications were demonstrated, letting the experimen-tal subjects undergo subchronic mild stressful situations.Suffice it to say, the morphological and immunocyto-chemical changes we have reported represent only a partof the numerous and complex anatomical and physiolog-ical abnormalities in adulthood as a result of maternalhyperthyroidism. However, this kind of animal modelsallow us to gain insight into normal development and toevaluate integrative aspects of the long-term influencesof neuroendocrine disorders during development.

ACKNOWLEDGMENTS

We thank Pablo G. Hofmann and Prof. RafaelBarrio for critical reading and insightful comments andLeyla Guadarrama, Guillermo Luna, Manuel Hernandez,and Fernando Carbajal for technical assistance.

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