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Maternal stress induces synaptic loss and developmental disabilities of offspring
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Pergamon
Int[ J[ Devl Neuroscience\ Vol[ 05\ No[ 2:3\ pp[ 198Ð105\ 0887 Þ 0887 ISDN[ Published by Elsevier Science Ltd All rights reserved[ Printed in Great Britain 9625Ð4637:87 ,08[99¦9[99
PII] S9625Ð4637"87#99917Ð7
MATERNAL STRESS INDUCES SYNAPTIC LOSS AND DEVELOPMENTAL DISABILITIES OF OFFSPRING ASKA HAYASHI\$ MASAAKI NAGAOKA\% KAZUO YAMADA\& YUKIO ICHITANI\& YOSHIAKI MIAKE% and NOBUO OKADO $ $Institute of Basic Medical Sciences\ University of Tsukuba\ Tsukuba Ibaraki 294\ Japan ^ %Department of Gynecology\ Nihon University\ School of Medicine\ Ohyaguchi\ Itabashi\ Tokyo 06\ Japan ^ &Institute of Psychology\ University of Tsukuba\ Tsukuba\ Ibaraki 294\ Japan "Received 18 December 0886 ^ accepted in revised form 05 March 0887# Abstract*Mild prenatal stress a}ects the serotonergic system in the hippocampus of rat o}spring[ Pregnant rats were daily exposed to mild stress treatments "consisting of crowding and saline injection# during days 04 to 10 of pregnancy[ Their o}spring were assessed by a series of biochemical\ histological and behavioral tests[ On 24 days after birth\ 4!hydroxytryptamine "4!HT# level was decreased by 06) "P ³ 9[94#\ whereas 4!hydroxyindolacetic acid "4!HIAA# level was increased by 07) "P ³ 9[94# in the o}spring of prenatally stressed rats[ The metabolic rate "4!HIAA:4!HT# was increased by 38) "P ³ 9[90#[ Synaptic density in the hippocampus of prenatally stressed o}spring was also decreased by 21) "P ³ 9[9990# on postnatal day 24[ There was no signi_cant group di}erence in the spatial learning acquisition test of the Morris water maze ^ however\ in the reversal task\ prenatally stressed 4!week old rats spent more time than control animals searching for the platform of the pool[ Escape latency in the cued test showed no signi_cant di}erence[ Together with data in our previous studies\ that have shown 4!HT to facilitate synapse formation and maintenance in the central nervous system\ synaptic loss is suggested to occur in relation to changes of 4!HT system in the hippocampus of prenatally stressed o}spring[ This may be associated with reported changes in behavior and learning ability in prenatally stressed o}spring[ Þ 0887 ISDN[ Published by Elsevier Science Ltd Key words] serotonin\ synapse\ hippocampus\ stress\ developmental disabilities[
Psychological stress during pregnancy has been predicted to induce behavioral aberrations in the children[ Retrospective studies on humans have reported a higher incidence of behavioral abnormalities in the children born from mothers who experienced psychological stress during pregnancy such as familial and marital discord\49 threat of impending war\18 or death of the husband[06 Animal studies have also supported this hypothesis by providing evidence that prenatally stressed o}spring show behavioral abnormalities including reduced propensity for social interac! tion\41 increased emotionality\43\44 increased locomotor response to novelty\02 and impaired sexual function[45 In addition\ studies on the learning ability of the o}spring of stressed mothers reveal impairments in the tasks ] maze learning ^2 reversal of a learning set on a T!maze ^38 and acquisition of an operant response[38 Although such environmental of epigenetic factors have been considered to induce a profound e}ect on the developing brain\ the underlying mechanism has long been unclear[ Regulation of the HPA axis has shown it to be a}ected by prenatal stress[ Many reports have shown a greater04\15\46 or more prolonged05\46 elevation of plasma corticosterone "B# after exposure to stress in prenatally stressed rats than in control rats[ Plasma B levels of prenatally stressed adult rats were reported to be higher even under basal conditions[04 In addition\ serotonin "4!HT# system has been shown to be changed by prenatal stress[27Ð32 Studies have shown the close relationship between the 4!HT system and the regulation of the HPA axis[03\29\21\37 4!HT is believed to play an important role in early brain development[07\08\10\47 Our previous studies have shown that 4!HT facilitates synapse formation and maintenance in the central nervous system[09\00\14\25\26 Considering the role of 4!HT for synapse formation and maintenance\ maternal stress changing the 4!HT system may a}ect synaptic density[ The question arises as to whether the 4!HT meditated synaptic density plays a role in the e}ect of prenatal stress on o}spring abnormal behavior[ Therefore\ to address
To whom all correspondence should be addressed[ Tel[ ] 9970 187 42 5850 ^ Fax ] 9970 187 42 5859 ^ E!mail ] nokadoÝmd[tsukudba[ac[jp 198
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this question we designed a study to investigate the long term e}ect of prenatal stress on the 4!HT system and the synaptic density of the hippocampus and hippocampus dependent spatial learning of the o}spring[ EXPERIMENTAL PROCEDURES Litters After mating\ female Wistar rats were either left undisturbed "mothers of control animals# or they were stressed during days 04Ð10 of pregnancy "mothers of prenatally stressed group#[ Stress treatments consisted of crowding "_ve pregnant rats in a 11×27×10 cm breeding cage# combined with once daily painful experiences by 9[90 ml of saline intramuscular injection as previously described[28 Preliminary study had provided evidence that this combination of crowding and daily saline injections elevated blood B levels by about 34)[ All litters were reduced to eight pups on the _rst day of birth to maintain uniform litter numbers and raised under standard laboratory conditions "room temperature 12>C\ lights on from 94 ] 99Ð08 ] 99 h#[ These experiments were permitted by the Community of Laboratory Animal Research Center in University of Tsukuba[ HPLC quanti_cation Thirty o}spring on 24 days after birth were used for each of the experimental and the control groups[ The hippocampus on the left side was immediately removed after the decapitation\ frozen with liquid nitrogen\ and stored in a deep freezer "−79>C# until the assay[ Concentration of 4!HT\ 4!hydroxyindoleacetic acid "4!HIAA# and noradrenaline "NA# was determined by using a high performance liquid chromatography with an electrochemical detection[ The hippocampus was homogenized in solution containing 499 ml of 9[1 M perchloric acid and containing 099 mM ethylenediaminetetraacetic acid "EDTA# and 149 ml isopreterenol[ Adequate separation of 4!HT\ 4! HIAA and NA was achieved using a mobile phase containing 9[929 M sodium acetate trihydrate\ 9[954 M citric acid monohydrate\ 29 mg 0!octanesulfanic acid sodium salt:l\ 4 mg EDTA = 1Na:l and 4) methanol at pH 2[9[ Identi_cation and quanti_cation of the biogenic amines was determined by comparison with known amounts of standards[ The statistical evaluation was carried out by grouped t!test or MannÐWhitney U!test[ Electron microscopy Four or _ve rats were used for each experimental and control group[ Animals were decapitated under deep anesthesia by pentobarbital on 24 days after birth[ The hippocampus in the right side was removed\ and immersed in a _xative containing phosphate!bu}ered 4) glutaraldehyde solution for 13 h[ One mm thick coronal sections of mid portion of the dorsal hippocampus was post_xed with phosphate!bu}ered 0) glutaraldehyde for 13 h[ After dehydration in graded series of ethanol solutions\ tissues were stained for 0 h in 0) phosphotungustic acid "EPTA#\ dehydrated in propylene oxide\ then embedded in Epon[ Four mm!thick sections were cut and stained with toluidine blue[ The density of synapses was examined in the superior part of stratum radiatum of CA2\ where the largest changes have been shown to occur in the synaptic density following perturbation of the 4! HT system[14 Semithin sections were trimmed to include only the CA2 region\ and re!embedded in Epon[ Ultrathin sections "79 nm# were cut using a diamond knife[ At least 19 electron micrographs were taken randomly at a primary magni_cation×09\999 "HITACHI H!599# from the superior part of the stratum radiatum[ Negatives were enlarged on printing papers\ and _nal magni_cation was×02\499[ The criteria for the identi_cation of synapses was the presence of postsynaptic density\ presynaptic dense projections "one or more#\ and intersynaptic cleft[ The statistical evaluation was carried out using grouped t!test or MannÐWhitney U!test[ Behavioral testing Behavioral testing was conducted in a Morris water maze[22\23 Sixteen animals were used for each control and prenatally stressed group[ The apparatus was a circular pool "049 cm diameter# _lled to a depth of 39 cm with water "1220>C#[ A translucent cylindrical Plexiglas platform "09 cm diameter# was submerged 0 cm below the water surface at one of four places\ 89 degrees apart\
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midway between the center and periphery of the pool[ Training in the water maze began when animals were 29 days old[ For the spatial learning acquisition test\ animals were subjected to a block of four trials per day for six consecutive days[ Each trial consisted for placing the rat in the water facing the wall of the pool at one of the four starting locations[ They were allowed to search for the platform for a maximum of 019 s\ and they could remain on the platform for 09 s[ After the spatial learning acquisition test\ animals were divided into two groups for the reversal task and the probe test[ The reversal task was carried out from the day after the last spatial learning acquisition test for three consecutive days "days 6Ð8#[ For the reversal task\ the platform was set on an opposite side from the place\ where the platform had been in the spatial learning acquisition test[ In any trial\ the starting point was randomly selected[ The swimming course was traced and measured\ and time spent from the starting point to the platform was registered[ The probe test was carried out on day 6[ To do this\ the platform was removed from the pool[ The rats were allowed to swim freely for 59 s[ The time spent in the quadrant where the removed platform had been was measured[ The cued test was carried out on the day after the probe test "day 7# and the day after the reversal task "day 09#[ The rats were required to locate the platform which had a black rim 9[4 cm and a black ~ag pole which was visible above the surface[ Each animal was given four trials starting from randomly selected starting points[ Di}erences between groups on the spatial learning acquisition test and reversal task were evaluated by analysis of variance "ANOVA# on repeated measures followed by Fisher|s protected least signi_cant di}erence "PLSD# test[ The data from the probe and cued test was analyzed by grouped t!test[
RESULTS Monoamine concentration The e}ect of maternal stress on 4!HT\ 4!HIAA and NA levels is summarized in Fig[ 0[ The e}ect from maternal stress was found on postnatal day 24 "Fig[ 0#[ The concentration of 4!HT had decreased by 06) "P ³ 9[94#\ whereas that of 4!HIAA had increased by 07) "P ³ 9[94# in the hippocampus of prenatally stressed pups compared to control[ The NA level showed no di}erence[ The metabolic rate of 4!HT "4!HIAA:4!HT# was increased by 38) "P ³ 9[90# in the prenatally stressed group "Fig[ 1#[
Fig[ 0[ The e}ect of prenatal stress on 4!HT\ 4!HIAA\ and NA concentration on postnatal day 24[ The values "mean2S[E[M[# are expressed as a percentage of control animals[ "P ³ 9[94#[
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Fig[ 1[ The e}ect of prenatal stress on 4!HT metabolism "4!HIAA:4!HT# on postnatal day 24[ The value "mean2S[E[M[# is expressed as a percentage of control animals[ "P ³ 9[90#[
Synaptic density The density of synapses in the hippocampus decreased by 21) "P ³ 9[9990# in prenatally stressed pups compared to control on 24 days after birth "Fig[ 2#[ Behavior Figure 3 illustrates the results for time in learning acquisition of the water maze task from day 29Ðday 25 after birth[ Although there was a signi_cant e}ect on day F"0\29# 002[16\ P ³ 9[9990\ there were no signi_cant e}ects on either group or the interaction between group and day[ On the reversal task "Fig[ 3b#\ there was no signi_cant main e}ect of group\ whereas an interaction between group and day\ F"1\17# 2[823\ P 9[92\ was signi_cant[ The Post!hoc test revealed that prenatally stressed o}springs showed signi_cant "P ³ 9[94# delay in time to reach the reversed platform on the _rst day of the reversal task "Day 6#[ Figure 3c illustrates the results of the probe test[ Both groups
Fig[ 2[ The e}ect of prenatal stress on synaptic density in the CA2 region of hippocampus on postnatal day 24[ The value "mean2S[E[M[# is expressed as a percentage of control animals[ "P ³ 9[9990#[
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Fig[ 3[ Escape latency "mean2S[E[M[# over 5!day period "3 trials:day# of the spatial learning acquisition test following prenatal stress "Fig[ 3a#[ Escape latency "mean2S[E[M[# over 2!day period "3 trials:day# of the reversal task following prenatal stress "Fig[ 3b#[ "P ³ 9[94# The percentage time "mean2S[E[M[# taken in the removed platform quadrant during the 59!s probe test "Fig[ 3c#[ Escape latency "mean2S[E[M[# of the probe test following prenatal stress "Fig[ 3d#[
showed a preference in the quadrant where the platform had been[ On the cued test\ no signi_cant di}erence between groups was found "Fig[ 3d#[ DISCUSSION The main _nding of the present study is that mild stress during late pregnancy e}ects the 4!HT system and the density of synapses in the hippocampus of the o}spring\ and that the o}spring showed developmental disabilities in the spatial learning and memory[ Prenatal stress on 4!HT concentration in the hippocampus The concentration of 4!HT and 4!HIAA in the hippocampus of the o}spring from stressed mothers was signi_cantly changed 24 days after birth[ Previous studies have reported that prenatal stress causes changes in 4!HT and 4!HIAA in several di}erent regions of the brain[27\34 Peters28 has shown that the rate of synthesis of ð03CŁ4!HT from L! ð03CŁ tryptophan in homogenates of the hippocampus was signi_cantly reduced in the o}spring 59 days after birth[ These results suggest that the decrease in the 4!HT level by prenatal stress shown
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in the present study is due to reduction of tryptophan hydroxylation in the hippocampus of prenatally stressed o}spring[ The hippocampal 4!HT metabolism was also changed in the prenatally stressed o}spring on 24 days after birth[ Changes in 4!HT metabolism caused by prenatal stress in the di}erent regions of the brain have been reported in previous studies[27\31\34 Previous reports have shown that maternal stress in late pregnancy e}ects fetal neuroendocrine and 4!HT system\32\36 and the placental bed artery[8 There are also reports that transient insult of the placental circulation has been shown to result in long!term alternation in 4!HT metabolism in the brain of the o}spring[7\20 From these reports it is likely that the changes in the 4!HT system in the brain of the o}spring is mediated by placental mechanisms a}ected by stress to the mother[ Prenatal effect on synaptic density in the hippocampus The synaptic density of the CA2 region of the hippocampus decreased in prenatally stressed rat on postnatal day 24[ A previous study has shown that 4!HT is necessary to maintain the normal number of synapses in the hippocampus of rat[14 Considering the role of 4!HT on synapses\ the decrease of synaptic density in the CA2 region may result from changes in the 4!HT system in the hippocampus by prenatal stress[ Prenatal effect on learning ability No signi_cant di}erences were seen in the spatial learning acquisition test and the probe test between the two groups[ This result is similar\ in a broad sense\ to Szuran|s report[40 In the reversal task\ prenatally stressed rats spent more time searching for the platform on the _rst day[ Although the platform is moved to the opposite quadrant\ the rats need only learn the new position of the platform[ Therefore\ the reversal task is the same as a spatial learning acquisition test in that the rats require spatial memory ^ however\ data obtained showed di}ering results in the spatial learning acquisition test and the reversal task[ Although spatial learning ability seems not a}ected by prenatal stress\ {rapidity| of acquiring the reversal task was in~uenced by prenatal stress[ The delay of escape latency in the _rst day of the reversal task may indicate that there is a disability\ in adapting the approach response to a new platform position\ in the prenatally stressed o}spring[ From the obtained results it seems possible that the prenatally stressed o}spring become less ~exible to change and more retarded in strategy shifting from the previous spatial learning acquisition test to the reversal task[ It is of interest that hippocampal lesioned animals show signi_cant de_cit in the reversal task[19\42 No e}ect of prenatal stress was seen on escape latency in the cued task[ Thus the delay seen in the _rst day of the reversal task\ may not result from motivational or sensorimotor factors[ The role of 4!HT in synaptic density and learning The present study has shown that stress to the mother rat in late gestation period decreased the level of 4!HT and the density of synapses in the hippocampus of pups on postnatal day 24[ From these results\ the role of 4!HT mediated synapse formation is supposed to exist in the cascade from epigenetic or environmental factors to developmental disabilities[ Matsukawa et al[14 have shown that the number of synapses in the hippocampus maintained by biogenic amines may be a crucial factor in the learning mechanism\ and that rats can learn and memorize without long term potentia! tion[ Therefore\ the normal number of synapses maintained by biogenic amines appears important to keep learning ability[ Many genetic diseases\ such as phenylketunuria\ Downs| syndrome\ and Rett syndrome are known to show mental retardation[ The noteworthy point is that the biogenic amine system also shows changes in these diseases[3\01\17\24\35 It is possible that synaptic density is also changed in the central nervous system of these genetic diseases\ considering that biogenic amines facilitate synapse formation[ The underlying mechanism of the developmental disabilities or mental retardation from either epigenetic or genetic factors has long been unknown\ but biogenic amine mediated synaptic density may play an important role in the cascade of mental retardation and developmental dis! abilities from both epigenetic and genetic factors[ The occurrence of synaptic overproduction was seen in the critical period of development\
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especially one year after birth[07\33 There also is a report that suggests this synaptic overproduction is not seen in the brain of Downs| syndrome patients[6 Therefore\ the synaptic overproduction may be a critical factor in developing the brain for normal function[ By improving environments or supplying biogenic amines in the critical period\ it may be possible to rescue the developing brain from mental retardation and developmental disabilities in the future[ Acknowled`ements*This research was supported by Grant!in!Aid for Scienti_c Research on Priority Areas on {{Functional Development of Neuronal Circuits|| "grant number\ 96168090# and Grant!in!Aid for Scienti_c Research "grant number\ 98379109# of the Ministry of Education\ Science\ Sports and Culture of Japan
REFERENCES 0[ Ader\ R[ and Plaut\ S[ M[\ E}ects of prenatal maternal handling and di}erential housing on o}spring emotionality\ plasma corticosterone levels\ and susceptibility to gastric erosions[ Pyscosom[ Med[\ 0857\ 29"2#\ 166Ð175[ 1[ Alonso\ J[\ Castellano\ M[ A[ and Rodriguez\ M[\ Behavioral lateralization in rats ] prenatal stress e}ects on sex di}erences[ Brain Res[\ 0880\ 428"0#\ 34Ð49[ 2[ Archer\ J[ E[ and Blackman\ D[ E[\ Prenatal psychological stress and o}spring behavior in rats and mice[ Dev[ Psycho[\ 0860\ 3"2#\ 082Ð137 3[ Bar!Peled\ O[\ Gross!Issero}\ R[\ Ben!Hur\ HJ[\ Hoskins\ I[\ Groner\ Y[ and Biegon\ A[\ Fetal human brain exhibits a prenatal peak in the density of serotonin 4!HT0A receptors[ Neurosci[ Lett[\ 0880\ 016"1#\ 062Ð065[ 4[ Barbazanges\ A[\ Piazza\ P[ V[\ Le Moal\ M[ and Maccari\ S[\ Maternal glucocorticoid secretion mediates long!term e}ects of prenatal stress[ J[ Neurosci[\ 0885\ 05"01#\ 2832Ð2838[ 5[ Barlow\ S[ M[\ Knight\ A[ F[ and Sullivan\ F[ M[\ Prevention by diazepam of adverse e}ects of maternal restraint stress on postnatal development and learning in the rat[ Teratolo`y\ 0868\ 08"0#\ 094Ð009[ 6[ Becker\ L[ E[\ Synaptic dysgenesis[ Can[ J[ Neurol[ Sci[\ 0880\ 07"1#\ 069Ð079[ 7[ Chanez\ C[\ Priam\ M[\ Flexor\ M[ A[\ Hamon\ M[\ Bourgoin\ S[\ Kordon\ C[ and MinKowski\ A[\ Long lasting e}ects of intrauterine growth retardation of 4!HT metabolism in the brain of developing rats[ Brain Res[\ 0870\ 196"1#\ 286Ð 397[ 8[ Chang\ C[ and Zhang\ J[\ The analysis of relationship between fetal stress and blood dynamics in fetal vessels and placental bed vessels[ Chi[ J[ Obstet[ Gynecol[\ "Chinese#\ 0885\ 20"0#\ 04Ð6[ 09[ Chen\ L[\ Hamaguchi\ K[\ Ogawa\ M[\ Hamada\ S[ and Okado\ N[\ pCPA reduces both monoaminergic a}erents and non!monoaminergic synapses in the cerebral cortex[ Neurosci[ Res[\ 0883\ 08"0#\ 000!004[ 00[ Chen\ L[\ Hamaguchi\ K[\ Hamada\ S[ and Okado\ N[\ Regionional di}erences of serotonin!mediated synaptic plasticity in the chicken spinal cord with development and aging[ J[ Neural Transplant[ plast[\ 0886\ 5"0#\ 30Ð37[ 01[ Curtius\ H[ C[\ Niederwieser\ A[\ Viscontini\ M[\ Leimbacher\ W[\ Wegmann\ H[\ Blehova\ B[\ Rey\ F[\ Schaub\ J[ and Schmidt\ H[\ Serotonin and dopamine synthesis in phenylketonuria[ Adv[ Exp[ Med[ Biol[\ 0870\ 022\ 166Ð180[ 02[ Deminiere\ J[ M[\ Piazza\ P[ V[\ Guegan\ G[\ Abrous\ N[\ Maccari\ S[\ Le Moal\ M[ and Simon\ H[\ Increased locomotor response to novelty and propensity to intravenous amphetamine self administration in adult o}spring of stressed mothers[ Brain Res[\ 0881\ 475\ 024Ð028[ 03[ Fisher\ L[ A[ and Brown\ M[ R[\ Central regulation of stress responses ] regulation of the autonomic nervous system and visceral function by corticotrophin releasing factor!30[ Bailliere|s Clin[ Endocr[ Metab[\ 0880\ 24Ð49[ 04[ Fride\ E[\ Dan\ Y[\ Feldon\ J[\ Halevy\ G[ and Weinstock\ M[\ E}ects of prenatal stress on vulnerability to stress in prepubertal and adult rats[ Phsyiol[ Behav[\ 0875\ 26\ 570Ð576[ 05[ Henry\ C[\ Kabbaj\ M[\ Simon\ H[\ Le!Moal\ M[ and Maccari\ S[\ Prenatal stress increases the hypothalamo!pituitary! adrenal axis response in young and adult rats[ J[ Neuroendocrinol[\ 0883\ 5"2#\ 230Ð234[ 06[ Huttenen\ M[ O[ and Niskanen\ P[\ Prenatal loss of father and psychiatric disorders[ Arch[ Gen[ Psychiat[\ 0867\ 24\ 318Ð320[ 07[ Huttenlocher\ P[ R[\ de Courten\ C[\ Garey\ L[ J[ and Van der Loos\ H[\ Synaptogenesis in human visual cortex!evidence for synapse elimination during normal development[ Neurosci[ Lett[\ 0871\ 22"2#\ 136Ð141[ 08[ Jacobs\ B[ L[ and Azmitia\ E[ C[\ Structure and function of the brain serotonin System[ Physiol[ Rev[\ 0881\ 61"0#\ 054Ð 118[ 19[ Kimble\ D[ P[ and Kimble\ R[ J[\ Hippocampectomy and response perseveration in the rat[ J[ Comp[ Physiol[ Psychol[\ 0854\ 59\ 363Ð365[ 10[ Lauder\ J[ M[ and Krebs\ H[\ Serotonin as a di}erentiation signal in early neurogenesis[ Dev[ Neurosci[\ 0867\ 0"0#\ 04Ð 29[ 11[ Lauder\ J[ M[\ Wallace\ J[ A[ and Krebs\ H[\ Roles for serotonin in neuroembryogenesis[ Adv[ Exp[ Med[ Biol[\ 0870\ 022\ 366Ð495[ 12[ Lauder\ J[ M[\ Wallace\ J[ A[\ Krebs\ H[\ Petrusz\ P[ and McCarthy\ K[\ In vivo and in vitro development of serotonergic neurons[ Brain Res[ Bull\ 0871\ 8"0Ð5#\ 594Ð514[ 13[ Maccari\ S[\ Piazza\ P[ V[\ Kabbaj\ M[\ Barbazanges\ A[\ Simon\ H[ and Le Moal\ M[\ Adoption reverses the long!term impairment in glucocorticoid feedback induced by prenatal stress[ J[ Neurosci[\ 0884\ 04\ 009Ð005[ 14[ Matsukawa\ M[\ Ogawa\ M[\ Nakadate\ K[\ Maeshima\ T[\ Ichitani\ Y[\ Kawai\ N[ and Okado\ N[\ Serotonin and acetylcholine are crucial to maintain hippocampal synapses and memory acquisition in rats[ Neurosci[ Lett[\ 0886\ 129"0#\ 02Ð05[ 15[ McCormick\ C[ M[\ Smythe\ J[ W[\ Sharma\ S[ and Meaney\ M[ J[\ Sex!speci_c e}ects of prenatal stress on hypothalamic! pituitary!adrenal responses to stress and brain glucocorticoid receptor density in adult rats[ Dev[ Brain Res[\ 0884\ 73\ 44Ð50[ 16[ McGivern\ R[ F[\ Poland\ R[ E[\ Taylor\ A[ N[\ Branch\ B[ J[ and Raum\ W[ J[\ Prenatal stress feminizes adult male saccharin preference and maze learning ] antagonism by propranolol[ Mono`r Neural Sci[\ 0875\ 01 061Ð067[
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17[ Mckean\ C[ M[\ The e}ects of high phenylalanine concentrations on serotonin and catecholamine metabolism in the human brain[ Brain Res[\ 0861\ 36"1#\ 358Ð365[ 18[ Meier\ A[\ Child psychiatric sequelae of maternal war stress[ Acta Psychiatr[ Scad[\ 0874\ 61\ 494Ð400[ 29[ Menderlson\ S[ D[ and McEwen\ B[ S[\ Autoradiographic analyses of the e}ects of adrenalectomy and corticosterone on 4!HT0A and 4!HT0B receptors in the dorsal hippocampus and cortex of the rat[ Neuroendocrinolo`y\ 0881\ 44\ 333Ð 349[ 20[ Minkowski\ A[\ Chanez\ C[\ Priam\ M[\ Flexor\ M[\ Hamon\ M[\ Bourgoin\ L[ and Kordon\ C[\ Long lasting e}ects of intrauterine malnutrition on neurotransmitters metabolism in the brain of developing rats[ Pro`[ Clin[ Biol[ Res[\ 0870\ 66\ 532Ð559[ 21[ Mitchell\ J[ B[\ Rowe[\ W[\ Boska\ P[ and Meaney\ M[ J[\ Serotonin regulates type II corticosteroid receptor binding in hippocampal cell culture[ J[ Neurosci[\ 0889\ 09\ 0634Ð0641[ 22[ Morris\ R[ G[\ Spatial localization does not require the presence of local cues[ Learn Motiv[\ 0870\ 01\ 128Ð159[ 23[ Morris\ R[ G[\ Developments of a water!maze procedure for studying spatial learning in the rat[ J[ Neurosci[ Methods[\ 0873\ 00\ 36Ð59[ 24[ Nielsen\ J[ B[\ Lou\ H[ C[ and Andresen\ J[\ Biochemical and clinical e}ects of tyrosine and tryptophan in the Rett syndrome[ Brain Develop[\ 0889\ 01"0#\ 032Ð036[ 25[ Niitsu\ Y[\ Hamada\ S[\ Hamaguchi\ K[\ Mikuni\ M[ and Okado\ N[\ Regulation of synapse density by 4!HT1A receptor agonist and antagonist in the spinal cord of chicken embryo[ Neurosci[ Lett[\ 0884\ 084"2#\ 048Ð051[ 26[ Okado\ N[\ Cheng\ L[\ Tanatsugu\ Y[\ Hamada\ S[ and Hamaguchi\ K[\ Synaptic loss following removal of serotoninergic _bers in newly hatched and adult chickens[ J[ Neurobiol[\ 0882\ 13"4#\ 576Ð587[ 27[ Peters\ D[ A[\ Prenatal stress ] e}ects on brain biogenic amine and plasma corticosterone levels[ Pharmacol[ Biochem[ Behav[\ 0871\ 06"3#\ 610Ð614[ 28[ Peters\ D[ A[\ Prenatal stress ] e}ect on development of rat brain serotonergic neurons[ Pharmacol[ Biochem[ Behav[\ 0875\ 13"4#\ 0266Ð0271[ 39[ Peters\ D[ A[\ Prenatal stress increases the behavioral response to serotonin agonists and alters open _eld behavior in the rat[ Pharmacol[ Biochem[ Behav[\ 0885\ 14"3#\ 762Ð766[ 30[ Peters\ D[ A[\ Both prenatal and postnatal factors contribute to the e}ects of maternal stress on o}spring behavior and central 4!hydroxytryptamine receptors in the rat[ Pharmacol[ Biochem[ Behav[\ 0877\ 29"2#\ 558Ð562[ 31[ Peters\ D[ A[\ E}ects of maternal stress during di}erent gestational periods of the serotonergic system in adult rat o}spring[ Pharmacol[ Biochem[ Behavr[\ 0877\ 20"3#\ 728Ð732[ 32[ Peters\ D[ A[\ Maternal stress increases fetal brain and neonatal cerebral cortex 4!hydroxytryptamin synthesis in rats ] a possible mechanism by which stress in~uences brain development[ Pharmacol[ Biochem[ Behav[\ 0889\ 24"3#\ 832Ð836[ 33[ Rakic\ P[\ Bourgeois\ J[ P[\ Eckenho}\ M[ F[\ Zecevic\ N[ and Goldman!Rakic\ P[ S[\ Concurrent over production of synapses in diverse regions of the primate cerebral cortex[ Science\ 0875\ 121\ 121Ð124[ 34[ Rezhikov\ A[ G[ and Nosenko\ N[ D[\ Early postnatal changes in sexual dimorphism of catecholamine and indoleamine content in the prenatally stressed rats[ Neuroscience[\ 0885\ 69"1#\ 436Ð440[ 35[ Riederer\ P[\ Weiser\ M[\ Wichart\ I[\ Schmidt\ B[\ Killian\ W[ and Rett\ A[\ Preliminary brain autopsy _ndings in progredient Rett syndrome[ Am[ J[ Med[ Genet[\ 0875\ 0\ 294Ð204[ 36[ Rohde\ W[\ Ohkawa\ T[\ Gotz\ F[\ Stahl[ F[\ Tonjes\ R[\ Takeshita\ S[\ Arakawa\ S[\ Kambegawa\ A[\ Arai\ K[ and Okinaga\ S[\ Sex!speci_c e}ects on the fetal neuroendocrine system during acute stress in late pregnancy of rat and the in~uence of simultaneous treatment by tyrosine[ Exp[ Clin[ Endocrinol[\ 0878\ 83"0Ð1#\ 12Ð31[ 37[ Siegel\ R[ A[\ Weidenfeld\ J[ and Chen\ M[\ Hippocampal cell nuclear binding of corticosterone following 4!6!dihy! droxytryptamine[ Molec[ Cell[ Endocr[\ 0872\ 20\ 142Ð248[ 38[ Smith\ B[ L[ Wills\ G[ and Naylor\ D[\ The e}ects of prenatal stress on rat o}springs| learning ability[ J[ Psychol[\ 0870\ 096\ 34Ð40[ 49[ Stott\ D[ H[\ Follow!up study from birth of the e}ects of prenatal stresses[ Dev[ Med[ Child[ Nerurol[\ 0862\ 04"5#\ 669Ð 676[ 40[ Szuran\ T[\ Zimmermann\ E[ and Welzl\ H[\ Water maze performance and hippocampal weight of prenatally stressed rats[ Behav[ Brain Res[\ 0883\ 54"1#\ 042Ð044[ 41[ Takahashi\ L[ K[ and Haglin\ N[ H[\ Prenatal stress potentiates stress!induced behavior and reduces propensity to play in juvenile rats[ Physiol[ Behav[\ 0881\ 40\ 208Ð212[ 42[ Thompson\ R[ and Langer\ S[ K[\ De_cits in position reversal learning following lesions of the limbic system[ J[ Comp[ Physiol[ Psychol[\ 0852\ 45\ 876Ð884[ 43[ Thompson\ W[ R[\ In~uence of prenatal maternal anxiety on emotionality in young rats[ Science\ 0846\ 04 587Ð588[ 44[ Wakshlak\[ A[ and Weinstock\ M[\ Neonatal handling reverses behavioral abnormalities induced in rats by prenatal stress[ Physiol[ Behav[\ 0889\ 37\ 178Ð181[ 45[ Ward\ I[ L[ and Reed\ J[\ Prenatal stress and prepuberal social rearing conditions interact to determine sexual behavior in male rats[ Behav[ Neurosci[\ 0874\ 88"1#\ 290Ð298[ 46[ Weinstock\ M[\ Matlina\ E[\ Maor\ G[ I[\ Rosen\ H[ and McEwen\ B[ S[\ Prenatal stress selectively alters the reactibility of the hypothalamic!pituitary!adrenal system in the female rat[ Brain Res[\ 0881\ 484\ 084Ð199[ 47[ Whitaker!Azmitia\ P[ M[ and Azmitia\ E[ C[\ Autoregulation of fetal serotonergic neuronal development ] role of high a.nity serotonin receptors[ Neurosci[ Lett[\ 0875\ 56"2#\ 296Ð201[
Pergamon
Int[ J[ Devl Neuroscience\ Vol[ 05\ No[ 2:3\ pp[ 198Ð105\ 0887 Þ 0887 ISDN[ Published by Elsevier Science Ltd All rights reserved[ Printed in Great Britain 9625Ð4637:87 ,08[99¦9[99
PII] S9625Ð4637"87#99917Ð7
MATERNAL STRESS INDUCES SYNAPTIC LOSS AND DEVELOPMENTAL DISABILITIES OF OFFSPRING ASKA HAYASHI\$ MASAAKI NAGAOKA\% KAZUO YAMADA\& YUKIO ICHITANI\& YOSHIAKI MIAKE% and NOBUO OKADO $ $Institute of Basic Medical Sciences\ University of Tsukuba\ Tsukuba Ibaraki 294\ Japan ^ %Department of Gynecology\ Nihon University\ School of Medicine\ Ohyaguchi\ Itabashi\ Tokyo 06\ Japan ^ &Institute of Psychology\ University of Tsukuba\ Tsukuba\ Ibaraki 294\ Japan "Received 18 December 0886 ^ accepted in revised form 05 March 0887# Abstract*Mild prenatal stress a}ects the serotonergic system in the hippocampus of rat o}spring[ Pregnant rats were daily exposed to mild stress treatments "consisting of crowding and saline injection# during days 04 to 10 of pregnancy[ Their o}spring were assessed by a series of biochemical\ histological and behavioral tests[ On 24 days after birth\ 4!hydroxytryptamine "4!HT# level was decreased by 06) "P ³ 9[94#\ whereas 4!hydroxyindolacetic acid "4!HIAA# level was increased by 07) "P ³ 9[94# in the o}spring of prenatally stressed rats[ The metabolic rate "4!HIAA:4!HT# was increased by 38) "P ³ 9[90#[ Synaptic density in the hippocampus of prenatally stressed o}spring was also decreased by 21) "P ³ 9[9990# on postnatal day 24[ There was no signi_cant group di}erence in the spatial learning acquisition test of the Morris water maze ^ however\ in the reversal task\ prenatally stressed 4!week old rats spent more time than control animals searching for the platform of the pool[ Escape latency in the cued test showed no signi_cant di}erence[ Together with data in our previous studies\ that have shown 4!HT to facilitate synapse formation and maintenance in the central nervous system\ synaptic loss is suggested to occur in relation to changes of 4!HT system in the hippocampus of prenatally stressed o}spring[ This may be associated with reported changes in behavior and learning ability in prenatally stressed o}spring[ Þ 0887 ISDN[ Published by Elsevier Science Ltd Key words] serotonin\ synapse\ hippocampus\ stress\ developmental disabilities[
Psychological stress during pregnancy has been predicted to induce behavioral aberrations in the children[ Retrospective studies on humans have reported a higher incidence of behavioral abnormalities in the children born from mothers who experienced psychological stress during pregnancy such as familial and marital discord\49 threat of impending war\18 or death of the husband[06 Animal studies have also supported this hypothesis by providing evidence that prenatally stressed o}spring show behavioral abnormalities including reduced propensity for social interac! tion\41 increased emotionality\43\44 increased locomotor response to novelty\02 and impaired sexual function[45 In addition\ studies on the learning ability of the o}spring of stressed mothers reveal impairments in the tasks ] maze learning ^2 reversal of a learning set on a T!maze ^38 and acquisition of an operant response[38 Although such environmental of epigenetic factors have been considered to induce a profound e}ect on the developing brain\ the underlying mechanism has long been unclear[ Regulation of the HPA axis has shown it to be a}ected by prenatal stress[ Many reports have shown a greater04\15\46 or more prolonged05\46 elevation of plasma corticosterone "B# after exposure to stress in prenatally stressed rats than in control rats[ Plasma B levels of prenatally stressed adult rats were reported to be higher even under basal conditions[04 In addition\ serotonin "4!HT# system has been shown to be changed by prenatal stress[27Ð32 Studies have shown the close relationship between the 4!HT system and the regulation of the HPA axis[03\29\21\37 4!HT is believed to play an important role in early brain development[07\08\10\47 Our previous studies have shown that 4!HT facilitates synapse formation and maintenance in the central nervous system[09\00\14\25\26 Considering the role of 4!HT for synapse formation and maintenance\ maternal stress changing the 4!HT system may a}ect synaptic density[ The question arises as to whether the 4!HT meditated synaptic density plays a role in the e}ect of prenatal stress on o}spring abnormal behavior[ Therefore\ to address
To whom all correspondence should be addressed[ Tel[ ] 9970 187 42 5850 ^ Fax ] 9970 187 42 5859 ^ E!mail ] nokadoÝmd[tsukudba[ac[jp 198
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this question we designed a study to investigate the long term e}ect of prenatal stress on the 4!HT system and the synaptic density of the hippocampus and hippocampus dependent spatial learning of the o}spring[ EXPERIMENTAL PROCEDURES Litters After mating\ female Wistar rats were either left undisturbed "mothers of control animals# or they were stressed during days 04Ð10 of pregnancy "mothers of prenatally stressed group#[ Stress treatments consisted of crowding "_ve pregnant rats in a 11×27×10 cm breeding cage# combined with once daily painful experiences by 9[90 ml of saline intramuscular injection as previously described[28 Preliminary study had provided evidence that this combination of crowding and daily saline injections elevated blood B levels by about 34)[ All litters were reduced to eight pups on the _rst day of birth to maintain uniform litter numbers and raised under standard laboratory conditions "room temperature 12>C\ lights on from 94 ] 99Ð08 ] 99 h#[ These experiments were permitted by the Community of Laboratory Animal Research Center in University of Tsukuba[ HPLC quanti_cation Thirty o}spring on 24 days after birth were used for each of the experimental and the control groups[ The hippocampus on the left side was immediately removed after the decapitation\ frozen with liquid nitrogen\ and stored in a deep freezer "−79>C# until the assay[ Concentration of 4!HT\ 4!hydroxyindoleacetic acid "4!HIAA# and noradrenaline "NA# was determined by using a high performance liquid chromatography with an electrochemical detection[ The hippocampus was homogenized in solution containing 499 ml of 9[1 M perchloric acid and containing 099 mM ethylenediaminetetraacetic acid "EDTA# and 149 ml isopreterenol[ Adequate separation of 4!HT\ 4! HIAA and NA was achieved using a mobile phase containing 9[929 M sodium acetate trihydrate\ 9[954 M citric acid monohydrate\ 29 mg 0!octanesulfanic acid sodium salt:l\ 4 mg EDTA = 1Na:l and 4) methanol at pH 2[9[ Identi_cation and quanti_cation of the biogenic amines was determined by comparison with known amounts of standards[ The statistical evaluation was carried out by grouped t!test or MannÐWhitney U!test[ Electron microscopy Four or _ve rats were used for each experimental and control group[ Animals were decapitated under deep anesthesia by pentobarbital on 24 days after birth[ The hippocampus in the right side was removed\ and immersed in a _xative containing phosphate!bu}ered 4) glutaraldehyde solution for 13 h[ One mm thick coronal sections of mid portion of the dorsal hippocampus was post_xed with phosphate!bu}ered 0) glutaraldehyde for 13 h[ After dehydration in graded series of ethanol solutions\ tissues were stained for 0 h in 0) phosphotungustic acid "EPTA#\ dehydrated in propylene oxide\ then embedded in Epon[ Four mm!thick sections were cut and stained with toluidine blue[ The density of synapses was examined in the superior part of stratum radiatum of CA2\ where the largest changes have been shown to occur in the synaptic density following perturbation of the 4! HT system[14 Semithin sections were trimmed to include only the CA2 region\ and re!embedded in Epon[ Ultrathin sections "79 nm# were cut using a diamond knife[ At least 19 electron micrographs were taken randomly at a primary magni_cation×09\999 "HITACHI H!599# from the superior part of the stratum radiatum[ Negatives were enlarged on printing papers\ and _nal magni_cation was×02\499[ The criteria for the identi_cation of synapses was the presence of postsynaptic density\ presynaptic dense projections "one or more#\ and intersynaptic cleft[ The statistical evaluation was carried out using grouped t!test or MannÐWhitney U!test[ Behavioral testing Behavioral testing was conducted in a Morris water maze[22\23 Sixteen animals were used for each control and prenatally stressed group[ The apparatus was a circular pool "049 cm diameter# _lled to a depth of 39 cm with water "1220>C#[ A translucent cylindrical Plexiglas platform "09 cm diameter# was submerged 0 cm below the water surface at one of four places\ 89 degrees apart\
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midway between the center and periphery of the pool[ Training in the water maze began when animals were 29 days old[ For the spatial learning acquisition test\ animals were subjected to a block of four trials per day for six consecutive days[ Each trial consisted for placing the rat in the water facing the wall of the pool at one of the four starting locations[ They were allowed to search for the platform for a maximum of 019 s\ and they could remain on the platform for 09 s[ After the spatial learning acquisition test\ animals were divided into two groups for the reversal task and the probe test[ The reversal task was carried out from the day after the last spatial learning acquisition test for three consecutive days "days 6Ð8#[ For the reversal task\ the platform was set on an opposite side from the place\ where the platform had been in the spatial learning acquisition test[ In any trial\ the starting point was randomly selected[ The swimming course was traced and measured\ and time spent from the starting point to the platform was registered[ The probe test was carried out on day 6[ To do this\ the platform was removed from the pool[ The rats were allowed to swim freely for 59 s[ The time spent in the quadrant where the removed platform had been was measured[ The cued test was carried out on the day after the probe test "day 7# and the day after the reversal task "day 09#[ The rats were required to locate the platform which had a black rim 9[4 cm and a black ~ag pole which was visible above the surface[ Each animal was given four trials starting from randomly selected starting points[ Di}erences between groups on the spatial learning acquisition test and reversal task were evaluated by analysis of variance "ANOVA# on repeated measures followed by Fisher|s protected least signi_cant di}erence "PLSD# test[ The data from the probe and cued test was analyzed by grouped t!test[
RESULTS Monoamine concentration The e}ect of maternal stress on 4!HT\ 4!HIAA and NA levels is summarized in Fig[ 0[ The e}ect from maternal stress was found on postnatal day 24 "Fig[ 0#[ The concentration of 4!HT had decreased by 06) "P ³ 9[94#\ whereas that of 4!HIAA had increased by 07) "P ³ 9[94# in the hippocampus of prenatally stressed pups compared to control[ The NA level showed no di}erence[ The metabolic rate of 4!HT "4!HIAA:4!HT# was increased by 38) "P ³ 9[90# in the prenatally stressed group "Fig[ 1#[
Fig[ 0[ The e}ect of prenatal stress on 4!HT\ 4!HIAA\ and NA concentration on postnatal day 24[ The values "mean2S[E[M[# are expressed as a percentage of control animals[ "P ³ 9[94#[
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Fig[ 1[ The e}ect of prenatal stress on 4!HT metabolism "4!HIAA:4!HT# on postnatal day 24[ The value "mean2S[E[M[# is expressed as a percentage of control animals[ "P ³ 9[90#[
Synaptic density The density of synapses in the hippocampus decreased by 21) "P ³ 9[9990# in prenatally stressed pups compared to control on 24 days after birth "Fig[ 2#[ Behavior Figure 3 illustrates the results for time in learning acquisition of the water maze task from day 29Ðday 25 after birth[ Although there was a signi_cant e}ect on day F"0\29# 002[16\ P ³ 9[9990\ there were no signi_cant e}ects on either group or the interaction between group and day[ On the reversal task "Fig[ 3b#\ there was no signi_cant main e}ect of group\ whereas an interaction between group and day\ F"1\17# 2[823\ P 9[92\ was signi_cant[ The Post!hoc test revealed that prenatally stressed o}springs showed signi_cant "P ³ 9[94# delay in time to reach the reversed platform on the _rst day of the reversal task "Day 6#[ Figure 3c illustrates the results of the probe test[ Both groups
Fig[ 2[ The e}ect of prenatal stress on synaptic density in the CA2 region of hippocampus on postnatal day 24[ The value "mean2S[E[M[# is expressed as a percentage of control animals[ "P ³ 9[9990#[
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Fig[ 3[ Escape latency "mean2S[E[M[# over 5!day period "3 trials:day# of the spatial learning acquisition test following prenatal stress "Fig[ 3a#[ Escape latency "mean2S[E[M[# over 2!day period "3 trials:day# of the reversal task following prenatal stress "Fig[ 3b#[ "P ³ 9[94# The percentage time "mean2S[E[M[# taken in the removed platform quadrant during the 59!s probe test "Fig[ 3c#[ Escape latency "mean2S[E[M[# of the probe test following prenatal stress "Fig[ 3d#[
showed a preference in the quadrant where the platform had been[ On the cued test\ no signi_cant di}erence between groups was found "Fig[ 3d#[ DISCUSSION The main _nding of the present study is that mild stress during late pregnancy e}ects the 4!HT system and the density of synapses in the hippocampus of the o}spring\ and that the o}spring showed developmental disabilities in the spatial learning and memory[ Prenatal stress on 4!HT concentration in the hippocampus The concentration of 4!HT and 4!HIAA in the hippocampus of the o}spring from stressed mothers was signi_cantly changed 24 days after birth[ Previous studies have reported that prenatal stress causes changes in 4!HT and 4!HIAA in several di}erent regions of the brain[27\34 Peters28 has shown that the rate of synthesis of ð03CŁ4!HT from L! ð03CŁ tryptophan in homogenates of the hippocampus was signi_cantly reduced in the o}spring 59 days after birth[ These results suggest that the decrease in the 4!HT level by prenatal stress shown
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in the present study is due to reduction of tryptophan hydroxylation in the hippocampus of prenatally stressed o}spring[ The hippocampal 4!HT metabolism was also changed in the prenatally stressed o}spring on 24 days after birth[ Changes in 4!HT metabolism caused by prenatal stress in the di}erent regions of the brain have been reported in previous studies[27\31\34 Previous reports have shown that maternal stress in late pregnancy e}ects fetal neuroendocrine and 4!HT system\32\36 and the placental bed artery[8 There are also reports that transient insult of the placental circulation has been shown to result in long!term alternation in 4!HT metabolism in the brain of the o}spring[7\20 From these reports it is likely that the changes in the 4!HT system in the brain of the o}spring is mediated by placental mechanisms a}ected by stress to the mother[ Prenatal effect on synaptic density in the hippocampus The synaptic density of the CA2 region of the hippocampus decreased in prenatally stressed rat on postnatal day 24[ A previous study has shown that 4!HT is necessary to maintain the normal number of synapses in the hippocampus of rat[14 Considering the role of 4!HT on synapses\ the decrease of synaptic density in the CA2 region may result from changes in the 4!HT system in the hippocampus by prenatal stress[ Prenatal effect on learning ability No signi_cant di}erences were seen in the spatial learning acquisition test and the probe test between the two groups[ This result is similar\ in a broad sense\ to Szuran|s report[40 In the reversal task\ prenatally stressed rats spent more time searching for the platform on the _rst day[ Although the platform is moved to the opposite quadrant\ the rats need only learn the new position of the platform[ Therefore\ the reversal task is the same as a spatial learning acquisition test in that the rats require spatial memory ^ however\ data obtained showed di}ering results in the spatial learning acquisition test and the reversal task[ Although spatial learning ability seems not a}ected by prenatal stress\ {rapidity| of acquiring the reversal task was in~uenced by prenatal stress[ The delay of escape latency in the _rst day of the reversal task may indicate that there is a disability\ in adapting the approach response to a new platform position\ in the prenatally stressed o}spring[ From the obtained results it seems possible that the prenatally stressed o}spring become less ~exible to change and more retarded in strategy shifting from the previous spatial learning acquisition test to the reversal task[ It is of interest that hippocampal lesioned animals show signi_cant de_cit in the reversal task[19\42 No e}ect of prenatal stress was seen on escape latency in the cued task[ Thus the delay seen in the _rst day of the reversal task\ may not result from motivational or sensorimotor factors[ The role of 4!HT in synaptic density and learning The present study has shown that stress to the mother rat in late gestation period decreased the level of 4!HT and the density of synapses in the hippocampus of pups on postnatal day 24[ From these results\ the role of 4!HT mediated synapse formation is supposed to exist in the cascade from epigenetic or environmental factors to developmental disabilities[ Matsukawa et al[14 have shown that the number of synapses in the hippocampus maintained by biogenic amines may be a crucial factor in the learning mechanism\ and that rats can learn and memorize without long term potentia! tion[ Therefore\ the normal number of synapses maintained by biogenic amines appears important to keep learning ability[ Many genetic diseases\ such as phenylketunuria\ Downs| syndrome\ and Rett syndrome are known to show mental retardation[ The noteworthy point is that the biogenic amine system also shows changes in these diseases[3\01\17\24\35 It is possible that synaptic density is also changed in the central nervous system of these genetic diseases\ considering that biogenic amines facilitate synapse formation[ The underlying mechanism of the developmental disabilities or mental retardation from either epigenetic or genetic factors has long been unknown\ but biogenic amine mediated synaptic density may play an important role in the cascade of mental retardation and developmental dis! abilities from both epigenetic and genetic factors[ The occurrence of synaptic overproduction was seen in the critical period of development\
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especially one year after birth[07\33 There also is a report that suggests this synaptic overproduction is not seen in the brain of Downs| syndrome patients[6 Therefore\ the synaptic overproduction may be a critical factor in developing the brain for normal function[ By improving environments or supplying biogenic amines in the critical period\ it may be possible to rescue the developing brain from mental retardation and developmental disabilities in the future[ Acknowled`ements*This research was supported by Grant!in!Aid for Scienti_c Research on Priority Areas on {{Functional Development of Neuronal Circuits|| "grant number\ 96168090# and Grant!in!Aid for Scienti_c Research "grant number\ 98379109# of the Ministry of Education\ Science\ Sports and Culture of Japan
REFERENCES 0[ Ader\ R[ and Plaut\ S[ M[\ E}ects of prenatal maternal handling and di}erential housing on o}spring emotionality\ plasma corticosterone levels\ and susceptibility to gastric erosions[ Pyscosom[ Med[\ 0857\ 29"2#\ 166Ð175[ 1[ Alonso\ J[\ Castellano\ M[ A[ and Rodriguez\ M[\ Behavioral lateralization in rats ] prenatal stress e}ects on sex di}erences[ Brain Res[\ 0880\ 428"0#\ 34Ð49[ 2[ Archer\ J[ E[ and Blackman\ D[ E[\ Prenatal psychological stress and o}spring behavior in rats and mice[ Dev[ Psycho[\ 0860\ 3"2#\ 082Ð137 3[ Bar!Peled\ O[\ Gross!Issero}\ R[\ Ben!Hur\ HJ[\ Hoskins\ I[\ Groner\ Y[ and Biegon\ A[\ Fetal human brain exhibits a prenatal peak in the density of serotonin 4!HT0A receptors[ Neurosci[ Lett[\ 0880\ 016"1#\ 062Ð065[ 4[ Barbazanges\ A[\ Piazza\ P[ V[\ Le Moal\ M[ and Maccari\ S[\ Maternal glucocorticoid secretion mediates long!term e}ects of prenatal stress[ J[ Neurosci[\ 0885\ 05"01#\ 2832Ð2838[ 5[ Barlow\ S[ M[\ Knight\ A[ F[ and Sullivan\ F[ M[\ Prevention by diazepam of adverse e}ects of maternal restraint stress on postnatal development and learning in the rat[ Teratolo`y\ 0868\ 08"0#\ 094Ð009[ 6[ Becker\ L[ E[\ Synaptic dysgenesis[ Can[ J[ Neurol[ Sci[\ 0880\ 07"1#\ 069Ð079[ 7[ Chanez\ C[\ Priam\ M[\ Flexor\ M[ A[\ Hamon\ M[\ Bourgoin\ S[\ Kordon\ C[ and MinKowski\ A[\ Long lasting e}ects of intrauterine growth retardation of 4!HT metabolism in the brain of developing rats[ Brain Res[\ 0870\ 196"1#\ 286Ð 397[ 8[ Chang\ C[ and Zhang\ J[\ The analysis of relationship between fetal stress and blood dynamics in fetal vessels and placental bed vessels[ Chi[ J[ Obstet[ Gynecol[\ "Chinese#\ 0885\ 20"0#\ 04Ð6[ 09[ Chen\ L[\ Hamaguchi\ K[\ Ogawa\ M[\ Hamada\ S[ and Okado\ N[\ pCPA reduces both monoaminergic a}erents and non!monoaminergic synapses in the cerebral cortex[ Neurosci[ Res[\ 0883\ 08"0#\ 000!004[ 00[ Chen\ L[\ Hamaguchi\ K[\ Hamada\ S[ and Okado\ N[\ Regionional di}erences of serotonin!mediated synaptic plasticity in the chicken spinal cord with development and aging[ J[ Neural Transplant[ plast[\ 0886\ 5"0#\ 30Ð37[ 01[ Curtius\ H[ C[\ Niederwieser\ A[\ Viscontini\ M[\ Leimbacher\ W[\ Wegmann\ H[\ Blehova\ B[\ Rey\ F[\ Schaub\ J[ and Schmidt\ H[\ Serotonin and dopamine synthesis in phenylketonuria[ Adv[ Exp[ Med[ Biol[\ 0870\ 022\ 166Ð180[ 02[ Deminiere\ J[ M[\ Piazza\ P[ V[\ Guegan\ G[\ Abrous\ N[\ Maccari\ S[\ Le Moal\ M[ and Simon\ H[\ Increased locomotor response to novelty and propensity to intravenous amphetamine self administration in adult o}spring of stressed mothers[ Brain Res[\ 0881\ 475\ 024Ð028[ 03[ Fisher\ L[ A[ and Brown\ M[ R[\ Central regulation of stress responses ] regulation of the autonomic nervous system and visceral function by corticotrophin releasing factor!30[ Bailliere|s Clin[ Endocr[ Metab[\ 0880\ 24Ð49[ 04[ Fride\ E[\ Dan\ Y[\ Feldon\ J[\ Halevy\ G[ and Weinstock\ M[\ E}ects of prenatal stress on vulnerability to stress in prepubertal and adult rats[ Phsyiol[ Behav[\ 0875\ 26\ 570Ð576[ 05[ Henry\ C[\ Kabbaj\ M[\ Simon\ H[\ Le!Moal\ M[ and Maccari\ S[\ Prenatal stress increases the hypothalamo!pituitary! adrenal axis response in young and adult rats[ J[ Neuroendocrinol[\ 0883\ 5"2#\ 230Ð234[ 06[ Huttenen\ M[ O[ and Niskanen\ P[\ Prenatal loss of father and psychiatric disorders[ Arch[ Gen[ Psychiat[\ 0867\ 24\ 318Ð320[ 07[ Huttenlocher\ P[ R[\ de Courten\ C[\ Garey\ L[ J[ and Van der Loos\ H[\ Synaptogenesis in human visual cortex!evidence for synapse elimination during normal development[ Neurosci[ Lett[\ 0871\ 22"2#\ 136Ð141[ 08[ Jacobs\ B[ L[ and Azmitia\ E[ C[\ Structure and function of the brain serotonin System[ Physiol[ Rev[\ 0881\ 61"0#\ 054Ð 118[ 19[ Kimble\ D[ P[ and Kimble\ R[ J[\ Hippocampectomy and response perseveration in the rat[ J[ Comp[ Physiol[ Psychol[\ 0854\ 59\ 363Ð365[ 10[ Lauder\ J[ M[ and Krebs\ H[\ Serotonin as a di}erentiation signal in early neurogenesis[ Dev[ Neurosci[\ 0867\ 0"0#\ 04Ð 29[ 11[ Lauder\ J[ M[\ Wallace\ J[ A[ and Krebs\ H[\ Roles for serotonin in neuroembryogenesis[ Adv[ Exp[ Med[ Biol[\ 0870\ 022\ 366Ð495[ 12[ Lauder\ J[ M[\ Wallace\ J[ A[\ Krebs\ H[\ Petrusz\ P[ and McCarthy\ K[\ In vivo and in vitro development of serotonergic neurons[ Brain Res[ Bull\ 0871\ 8"0Ð5#\ 594Ð514[ 13[ Maccari\ S[\ Piazza\ P[ V[\ Kabbaj\ M[\ Barbazanges\ A[\ Simon\ H[ and Le Moal\ M[\ Adoption reverses the long!term impairment in glucocorticoid feedback induced by prenatal stress[ J[ Neurosci[\ 0884\ 04\ 009Ð005[ 14[ Matsukawa\ M[\ Ogawa\ M[\ Nakadate\ K[\ Maeshima\ T[\ Ichitani\ Y[\ Kawai\ N[ and Okado\ N[\ Serotonin and acetylcholine are crucial to maintain hippocampal synapses and memory acquisition in rats[ Neurosci[ Lett[\ 0886\ 129"0#\ 02Ð05[ 15[ McCormick\ C[ M[\ Smythe\ J[ W[\ Sharma\ S[ and Meaney\ M[ J[\ Sex!speci_c e}ects of prenatal stress on hypothalamic! pituitary!adrenal responses to stress and brain glucocorticoid receptor density in adult rats[ Dev[ Brain Res[\ 0884\ 73\ 44Ð50[ 16[ McGivern\ R[ F[\ Poland\ R[ E[\ Taylor\ A[ N[\ Branch\ B[ J[ and Raum\ W[ J[\ Prenatal stress feminizes adult male saccharin preference and maze learning ] antagonism by propranolol[ Mono`r Neural Sci[\ 0875\ 01 061Ð067[
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A[ Hayashi et al[
17[ Mckean\ C[ M[\ The e}ects of high phenylalanine concentrations on serotonin and catecholamine metabolism in the human brain[ Brain Res[\ 0861\ 36"1#\ 358Ð365[ 18[ Meier\ A[\ Child psychiatric sequelae of maternal war stress[ Acta Psychiatr[ Scad[\ 0874\ 61\ 494Ð400[ 29[ Menderlson\ S[ D[ and McEwen\ B[ S[\ Autoradiographic analyses of the e}ects of adrenalectomy and corticosterone on 4!HT0A and 4!HT0B receptors in the dorsal hippocampus and cortex of the rat[ Neuroendocrinolo`y\ 0881\ 44\ 333Ð 349[ 20[ Minkowski\ A[\ Chanez\ C[\ Priam\ M[\ Flexor\ M[\ Hamon\ M[\ Bourgoin\ L[ and Kordon\ C[\ Long lasting e}ects of intrauterine malnutrition on neurotransmitters metabolism in the brain of developing rats[ Pro`[ Clin[ Biol[ Res[\ 0870\ 66\ 532Ð559[ 21[ Mitchell\ J[ B[\ Rowe[\ W[\ Boska\ P[ and Meaney\ M[ J[\ Serotonin regulates type II corticosteroid receptor binding in hippocampal cell culture[ J[ Neurosci[\ 0889\ 09\ 0634Ð0641[ 22[ Morris\ R[ G[\ Spatial localization does not require the presence of local cues[ Learn Motiv[\ 0870\ 01\ 128Ð159[ 23[ Morris\ R[ G[\ Developments of a water!maze procedure for studying spatial learning in the rat[ J[ Neurosci[ Methods[\ 0873\ 00\ 36Ð59[ 24[ Nielsen\ J[ B[\ Lou\ H[ C[ and Andresen\ J[\ Biochemical and clinical e}ects of tyrosine and tryptophan in the Rett syndrome[ Brain Develop[\ 0889\ 01"0#\ 032Ð036[ 25[ Niitsu\ Y[\ Hamada\ S[\ Hamaguchi\ K[\ Mikuni\ M[ and Okado\ N[\ Regulation of synapse density by 4!HT1A receptor agonist and antagonist in the spinal cord of chicken embryo[ Neurosci[ Lett[\ 0884\ 084"2#\ 048Ð051[ 26[ Okado\ N[\ Cheng\ L[\ Tanatsugu\ Y[\ Hamada\ S[ and Hamaguchi\ K[\ Synaptic loss following removal of serotoninergic _bers in newly hatched and adult chickens[ J[ Neurobiol[\ 0882\ 13"4#\ 576Ð587[ 27[ Peters\ D[ A[\ Prenatal stress ] e}ects on brain biogenic amine and plasma corticosterone levels[ Pharmacol[ Biochem[ Behav[\ 0871\ 06"3#\ 610Ð614[ 28[ Peters\ D[ A[\ Prenatal stress ] e}ect on development of rat brain serotonergic neurons[ Pharmacol[ Biochem[ Behav[\ 0875\ 13"4#\ 0266Ð0271[ 39[ Peters\ D[ A[\ Prenatal stress increases the behavioral response to serotonin agonists and alters open _eld behavior in the rat[ Pharmacol[ Biochem[ Behav[\ 0885\ 14"3#\ 762Ð766[ 30[ Peters\ D[ A[\ Both prenatal and postnatal factors contribute to the e}ects of maternal stress on o}spring behavior and central 4!hydroxytryptamine receptors in the rat[ Pharmacol[ Biochem[ Behav[\ 0877\ 29"2#\ 558Ð562[ 31[ Peters\ D[ A[\ E}ects of maternal stress during di}erent gestational periods of the serotonergic system in adult rat o}spring[ Pharmacol[ Biochem[ Behavr[\ 0877\ 20"3#\ 728Ð732[ 32[ Peters\ D[ A[\ Maternal stress increases fetal brain and neonatal cerebral cortex 4!hydroxytryptamin synthesis in rats ] a possible mechanism by which stress in~uences brain development[ Pharmacol[ Biochem[ Behav[\ 0889\ 24"3#\ 832Ð836[ 33[ Rakic\ P[\ Bourgeois\ J[ P[\ Eckenho}\ M[ F[\ Zecevic\ N[ and Goldman!Rakic\ P[ S[\ Concurrent over production of synapses in diverse regions of the primate cerebral cortex[ Science\ 0875\ 121\ 121Ð124[ 34[ Rezhikov\ A[ G[ and Nosenko\ N[ D[\ Early postnatal changes in sexual dimorphism of catecholamine and indoleamine content in the prenatally stressed rats[ Neuroscience[\ 0885\ 69"1#\ 436Ð440[ 35[ Riederer\ P[\ Weiser\ M[\ Wichart\ I[\ Schmidt\ B[\ Killian\ W[ and Rett\ A[\ Preliminary brain autopsy _ndings in progredient Rett syndrome[ Am[ J[ Med[ Genet[\ 0875\ 0\ 294Ð204[ 36[ Rohde\ W[\ Ohkawa\ T[\ Gotz\ F[\ Stahl[ F[\ Tonjes\ R[\ Takeshita\ S[\ Arakawa\ S[\ Kambegawa\ A[\ Arai\ K[ and Okinaga\ S[\ Sex!speci_c e}ects on the fetal neuroendocrine system during acute stress in late pregnancy of rat and the in~uence of simultaneous treatment by tyrosine[ Exp[ Clin[ Endocrinol[\ 0878\ 83"0Ð1#\ 12Ð31[ 37[ Siegel\ R[ A[\ Weidenfeld\ J[ and Chen\ M[\ Hippocampal cell nuclear binding of corticosterone following 4!6!dihy! droxytryptamine[ Molec[ Cell[ Endocr[\ 0872\ 20\ 142Ð248[ 38[ Smith\ B[ L[ Wills\ G[ and Naylor\ D[\ The e}ects of prenatal stress on rat o}springs| learning ability[ J[ Psychol[\ 0870\ 096\ 34Ð40[ 49[ Stott\ D[ H[\ Follow!up study from birth of the e}ects of prenatal stresses[ Dev[ Med[ Child[ Nerurol[\ 0862\ 04"5#\ 669Ð 676[ 40[ Szuran\ T[\ Zimmermann\ E[ and Welzl\ H[\ Water maze performance and hippocampal weight of prenatally stressed rats[ Behav[ Brain Res[\ 0883\ 54"1#\ 042Ð044[ 41[ Takahashi\ L[ K[ and Haglin\ N[ H[\ Prenatal stress potentiates stress!induced behavior and reduces propensity to play in juvenile rats[ Physiol[ Behav[\ 0881\ 40\ 208Ð212[ 42[ Thompson\ R[ and Langer\ S[ K[\ De_cits in position reversal learning following lesions of the limbic system[ J[ Comp[ Physiol[ Psychol[\ 0852\ 45\ 876Ð884[ 43[ Thompson\ W[ R[\ In~uence of prenatal maternal anxiety on emotionality in young rats[ Science\ 0846\ 04 587Ð588[ 44[ Wakshlak\[ A[ and Weinstock\ M[\ Neonatal handling reverses behavioral abnormalities induced in rats by prenatal stress[ Physiol[ Behav[\ 0889\ 37\ 178Ð181[ 45[ Ward\ I[ L[ and Reed\ J[\ Prenatal stress and prepuberal social rearing conditions interact to determine sexual behavior in male rats[ Behav[ Neurosci[\ 0874\ 88"1#\ 290Ð298[ 46[ Weinstock\ M[\ Matlina\ E[\ Maor\ G[ I[\ Rosen\ H[ and McEwen\ B[ S[\ Prenatal stress selectively alters the reactibility of the hypothalamic!pituitary!adrenal system in the female rat[ Brain Res[\ 0881\ 484\ 084Ð199[ 47[ Whitaker!Azmitia\ P[ M[ and Azmitia\ E[ C[\ Autoregulation of fetal serotonergic neuronal development ] role of high a.nity serotonin receptors[ Neurosci[ Lett[\ 0875\ 56"2#\ 296Ð201[