Effect of neonatal exposure to monosodium l-glutamate on regional GABA release during postnatal developmentfn1

NEUROCHEMISTRY International Neurochem[ Int[ 22 "0887# 106Ð121

E}ect of neonatal exposure to monosodium L!glutamate on regional GABA release during postnatal development C[ Beas!Zaratea\$\ M[ Y[ Sanchez!Ru(za\ M[ E[ Uren½a!Guerreroa\ A[ Feria!Velascob a

Lab[ de Neuroqu(mica Div[ de Patol[ Expl[\ C[I[B[O[\ I[M[S[S[\ Depto[ de Biol[ Cel[ y Molec[\ Div[ de Cs[ Biol[ y Ambs[\ C[U[C[B[A[\ U[ de G[\ Guadalajara\ Jalisco\ Mexico b Division de Patolo`(a y Biotecnolo`(a Ambiental\ CIATEJ[ Guadalajara\ Jalisco\ Mexico Received 0 June 0886^ revised 10 December 0886^ received for publication 3 March 0887

Abstract Monosodium L!glutamate "MSG# causes neuronal lesions in certain brain regions when systemically given to young animals[ Also\ when glutamate "Glu# builds up in the intersynaptic space\ it induces neuroexcitatory and neurocytotoxic e}ects\ events mediated by several Glu receptors[ Some of these receptors such as NMDA and AMPA receptors are present in the very earliest developmental stages of the central nervous system and play a major role in neuronal plasticity during synaptogenesis[ In this paper\ the GABAergic system vulnerability was determined in terms of ð2HŁ!GABA release during postnatal development[ ð2HŁ!GABA release on days 03\ 10\ 29\ and 59 days after birth was assessed for the cerebral cortex "CC#\ hippocampus "Hp# and striatum "S# in rats perinatally treated at days 0\ 2\ 4\ and 6 after birth with MSG[ The results show a major decrease in baseline ð2HŁ!GABA release in the CC "29 and 59 days after birth# and the Hp "beginning day 10 after birth# vs the control groups ðintact rats and rats given a NaCl solution equimolar to that of MSG "eqNaCl#Ł while in the S baseline release remained unchanged[ Stimulated ð2HŁ!GABA release was decreased in the CC on days 03 and 10 after birth and signi_cantly increased on day 59 after birth vs the controls[ In the Hp\ a decrease was seen on days 03\ 10\ and 59 after birth vs the controls while stimulated ð2HŁ!GABA release was decreased in the S vs the controls at all ages studied[ No signi_cant di}erences in stimulated ð2HŁ!GABA release were found between the intact group and the group treated with eqNaCl on days 29 and 59 after birth[ Results show that CC\ Hp and S GABAergic neurones are a major target for the e}ect of perinatally given MSG and suggest a possible decrease in the number of Hp GABAergic neurones while these results in CC and S suggest a modi_ed neuronal plasticity[ NMDA receptor and calcium involvement are discussed as signi_cant mediators of these events[ Þ 0887 Elsevier Science Ltd[ All rights reserved[ Key words] GABA^ Release^ Development^ Glutamate^ Cerebral Cortex^ Hippocampus^ Striatum

0[ Introduction Glutamic acid "Glu# given systemically "subcutaneously or intraperitoneally# as a monosodium salt\ at a dose of 3 mg:g body weight\ to newborn rats causes lesions to the retina\ circumventricular organs and other brain regions "Lucas and Newhouse 0846^ Olney\ 0860^ Rothman and Olney\ 0875# while giving the same Glu dose to adult rats results in general tonic clonic convulsive crises[ For this reason\ it has been used in experimental models to study some aspects of epilepsy "Robinson and Coyle\ 0876^ Beas!Zarate et al[\ 0878#[ In the central nervous system "CNS# of mammals\ Glu is mainly found in cortical inter! neurones "Streit\ 0874^ Gui}rida and Rustioni\ 0878# and  Due to circumstances beyond the Publisher|s control\ this article appears without the authors| corrections[ $ Corresponding author[ 9086Ð9075:87 ,08[99 Þ 0887 Elsevier Science Ltd[ All rights reserved PII] S 9 0 8 6 Ð 9 0 7 5 " 8 7 # 9 9 9 9 1 Ð 2

cortical a}erences reaching the corpus striatum "McGeer et al[\ 0866^ Carroll and Wong!Riley\ 0876#\ the molec! ular layer of the dentate gyrus "Sandoval and Cotman 0867# and mossy _bers\ among other brain regions "Terrian et al[\ 0877#[ Interest has increased over the last few decades to study the role of Glu in the CNS\ especially its neuroexcitatory and neurocytotoxic e}ects when large quantities build up in the intersynaptic space of several brain regions "Lucas and Newhouse 0846^ Olney\ 0858b^ Olney\ 0868#[ There are several studies showing the excitatory and cytotoxic e}ects of Glu by excessive receptor activation "Choi and Rothman\ 0889^ Olney\ 0867# both in vitro "Olney et al[\ 0876^ Zeevalk et al[\ 0878^ Zeevalk and Nicklas\ 0889^ Romano et al[\ 0884# and in vivo "Olney\ 0858b^ Ikinomidou et al[\ 0878^ Freese et al[\ 0889#[ Thus\ various Glu receptor subtypes have been involved in excitatory and cytotoxic Glu!induced neuronal death

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"Sillito\ 0864^ Tsumoto et al[\ 0868# especially the NMDA type "Tsumoto et al[\ 0868# and kainate type "McGeer et al[\ 0867^ Koh et al[\ 0889^ Choi\ 0881^ Sheardown et al[\ 0882# Glu receptors[ It has also been described that the neuronal response during the excitatory and cytotoxic processes depends on the Glu receptor subtype being activated and the type of biochemical and morphological alterations which also vary depending on the brain region under study[ Another signi_cant factor in~uencing the Glu excitatory and cytotoxic processes when used in in vivo systems is the age of the animal while in in vitro systems\ the time and cell culture conditions are the main factors to be considered "Rothman\ 0873^ Frandsen and Schousboe\ 0889^ Choi\ 0877^ Chan et al[\ 0884#[ Thus\ Glu neuroexcitatory and neurocytotoxic e}ects\ when given systemically\ are more obvious during the _rst 09 days after birth "Olney\ 0858a^ Ikinomidou et al[\ 0878#[ Accordingly\ recent studies in rats have shown that Glu receptors are present in the CNS very early in the devel! opment "Insel et al[\ 0889^ Miller et al[\ 0889^ Pellegrini! Giampietro et al[\ 0880# signi_cantly involving NMDA and AMPA type receptors in synaptic reorganization and remodelling over the _rst 2 weeks of age "Franceschetti et al[\ 0882^ Scheetz and Constantine!Paton\ 0883#[ Hence\ an overactivation of these receptors during the _rst 09 days of age could be a de_nite factor in estab! lishing interneuronal communication and modifying the synaptic physiology[ The purpose of this paper was to evaluate the release of exogeneous g!aminobutyric acid "GABA# in slices of di}erent brain regions with glu! tamatergic innervation such as the cerebral cortex\ stri! atum and hippocampus of rats treated with monosodium L!glutamate "MSG# during the _rst week of age[ This\ to learn about the vulnerability of the GABAergic system to Glu excitatory and cytotoxic e}ects and about neuronal responsiveness of these regions in addition to the possi! bility to implement a model to study some neuro! degenerative processes induced in an in vivo system[ 1[ Materials and methods 1[0[ Materials Amino!oxyacetic acid and monosodium L!glutamate "MSG# were purchased from Sigma Chemical Co[\ St Louis MO\ U[S[A[ g!ð1\2!2H"N#Ł!aminobutyric acid "ð2HŁ!GABA# "spec[ act[ 25[7 Ci:mml# was purchased from New England Nuclear\ Du Pont Co[\ Wilmington\ DE\ U[S[A[ All other chemicals used were commercially obtained and were the highest purity available[ 1[1[ Animals Pregnant Wistar rats were used and kept in optimal environmental conditions\ i[e[ free access to water and

food\ with 01×01 h light!darkness cycles\ temperatures ranging between 12>C and 14>C\ and in separate cages[ On the day of birth\ all litters were adjusted to 7 o}spring per female[ Following Dada et al[\ "0873# and Clough et al[\ "0875#\ o}spring were given subcutaneously 3 mg:g of body weight of either a saline solution "internal control group# or a sodium chloride solution equimolar to that of MSG solution "eqNaCl# "0[27 mg:g# on days 0\ 2\ 4\ and 6 of postnatal age[ There was also a group of un! treated animals "intact# used as controls[ All treated and intact animals were killed by decapitation on days 03\ 10\ 29\ and 59 after birth[ Several brain regions "cerebral cortex\ hippocampus\ and striatum# were then dissected out at a 3>C temperature and tissue slices were made for ð2HŁ!GABA release studies[ All the animals in the di}erent groups were from the same litters and from parallel litters[

1[2[ ð2HŁ!GABA release studies Release studies were conducted following Beas!Zarate et al[ "0878#[ To measure the baseline ð2HŁ!GABA release\ a Krebs!bicarbonate medium was used[ It is made of 004 mM NaCl\ 2 mM KCl\ 0[1 mM MgSO3\ 0[1 mM NaH1PO3\ 14 mM NaHCO2\ 0[4 mM CaCl1\ 09 mM glucose\ 9[90 mM aminooxyacetic acid\ pH adjusted to 6[3 with a CO1ÐO1 mixture "4Ð84)#[ Tissue slices "14 mg# were preincubated for 09 min in 0 ml of incubation medium at 26>C[ ð2HŁ!GABA was added at a 9[90 mM concentration[ Incubation continued for an additional 19 min and at the end of this time the samples were trans! ferred to a Brandel SP!95 perfusion system[ The tissue was then washed in a Krebs!bicarbonate solution for 4 min at a 0[1 ml:min ~ow rate and then washed again for another 4 min at a 9[4 ml:min ~ow rate[ For each region\ two parallel and simultaneous per! fusion pathways were used[ One of these was to derive baseline release values during the collection of 09 inde! pendent fractions\ at 4 min intervals and a 9[4 ml:min ~ow rate[ The other pathway was to collect the _rst 2 fractions to derive the baseline release value and with fraction number 3\ the medium having 42 mM KCl was changed to induce GABA stimulated release[ Under these conditions\ the next 3 fractions "fractions 3 through 6# were collected and in fraction number 7\ the Krebs!bicar! bonate solution with 2 mM KCl was again used to collect the last 2 fractions "7 through 09#[ At the end of collections\ 4 ml of scintillation liquid "Ready safe cocktail by Beckman# were added and radio! activity was quantitated with a Beckman LS!5999 SE spectrometer[ Release studies of all groups were conducted in the same system and at the same time[ The results from the animal groups given saline solution compared to those of intact animals of same ages were no di}erent from

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each other and both results were put together to set the corresponding control value[ Results were stated as a percentage of ð2HŁ!GABA released over the total ð2HŁ!GABA in the tissue[ Each _gure represents the average value2the standard devi! ation of the mean[ 1[3[ Statistical analysis Statistical signi_cance in the average values of the groups was calculated from an analysis of variance "ANOVA# and a Students t test using the SPSS computer software[

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2[2[ Striatal There were no signi_cant di}erences in ð2HŁ!GABA spon! taneous release in striatal slices in any ages studied because of either the MSG or eqNaCl tratment vs the intact group "Fig[ 6"a#Ð"d##[ In the MSG group\ however\ a major decrease in both ð2HŁ!GABA stimulated release and over~ow was seen at all ages studied vs the intact group and the eqNaCl group "Figs 7"a#Ð"d# and 8"a#Ð "d##[ By giving eqNaCl a decrease in ð2HŁ!GABA release and over~ow was also seen on days 03\ 10\ and 59 after birth vs the control group[ This\ although at a lower rate than in the MSG group\ was statistically signi_cant "Figs 7"a#\"b#\"d# and 8"a#\"b#\"d##[

2[ Results 2[0[ Cerebral cortex Results showed that giving MSG to a rat at an early age induced a signi_cant decrease in spontaneous ð2HŁ! GABA release beginning day 03 after birth[ This con! tinued until adulthood vs control groups treated with eqNaCl "Fig[ 0#[ Also\ the eqNaCl group showed no signi_cant changes in spontaneous ð2HŁ!GABA release vs the control group at all ages studied "Fig[ 0#[ On the other hand\ results also showed a major decrease in stimulated release and a ð2HŁ!GABA over~ow in the MSG group on days 03 and 10 after birth vs the control groups "Figs 1"a#\"b# and 2"a#\"b## while on day 59 after birth\ a signi_cant increase in ð2HŁ!GABA over! ~ow and stimulated release were seen in the MSG group vs the intact and eqNaCl groups "Figs 1"d# and 2"d##[ The eqNaCl group showed only a decrease in ð2HŁ!GABA over~ow and stimulated release on days 03 and 10 after birth vs the intact group "Figs 1"a#\"b# and 2"a#\"b## while no signi_cant changes were seen on days 29 and 59 after birth vs the intact group "Figs 1"c#\"d# and 2"c#\"d##[ 2[1[ Hippocampus In the hippocampus\ spontaneous ð2HŁ!GABA release was signi_cantly decreased in the MSG group at all ages studied vs the control groups "Fig[ 3"a#Ð"d##[ EqNaCl decreased signi_cantly the spontaneous ð2HŁ!GABA release only on days 10 after birth vs the intact group "Fig[ 3"b##[ On the other hand\ both ð2HŁ!GABA stimulated release and over~ow in hippocampus slices were signi_cantly decreased by the MSG perinatal treatment on days 03\ 10\ and 59 after birth vs the intact group and the eqNaCl group "Figs 4"a#\"b#\"d# and 5"a#\"b#\"d##[ Likewise\ there was a statistically signi_cant decrease in ð2HŁ!GABA stimulated release and over~ow in the eqNaCl group vs the intact group at 03\ 10 days after birth "Figs 4"a#\"b#\"d# and 5"a#\"b#\"d##[

3[ Discussion Over the last 19 years\ di}erent neurotoxins have been widely used as tools to study the mechanisms involved in several neurodegenerative diseases\ especially those related to excitatory aminoacids such as Glu "Olney\ 0863^ Silverstein et al[\ 0876^ Erdo and Michler\ 0889^ Frandsen and Schousboe\ 0889#[ This aminoacid is thought to be the most important excitatory neuro! transmitter of the CNS and receptor activation is appar! ently involved not only with an increased synaptic e.ciency but also with excitatory and cytotoxic e}ects as well as with neuronal death "Scheetz and Constantine! Paton\ 0883#[ Thus\ giving Glu systemically to newborn rodents results in extensive neuronal damage and decrease of certain markers of di}erent neurotransmitters in several CNS areas "Nemero} et al[\ 0866^ Dawson and Annau\ 0872#[ Accordingly\ selective vulnerability in some neurotransmission systems such as the GABAergic system has been proposed for several neurological dis! eases "McGeer et al[\ 0866^ Spokes\ 0870^ Streit et al[\ 0874^ Carroll and Wong!Riley\ 0876^ Gui}rida and Rus! tioni 0878^ Sourkes\ 0878^ Paredes and Agmo\ 0881#[ This paper evaluated the susceptibility of the GABAergic system in terms of GABA release in various brain regions during the postnatal development of rats after being given Glu as monosodium salt during the _rst week of life[ The results suggest that GABAergic neurons in the regions studied are a major target for the Glu e}ect[ And in the cerebral cortex GABA baseline release is not signi_cantly modi_ed during the _rst three weeks of age "Fig[ 0#[ On the other hand\ beginning day 29 after birth\ a decreased baseline GABA release "Fig[ 0# and an appar! ently increased stimulated release are seen on day 59 after birth "Figs 1 and 2#[ This suggests a possible decrease in the number of GABA!releasing neurons and greater e.ciency in the prevailing GABAergic cells which could be a neuronal plasticity phenomenon induced by Glu receptor overstimulation in early CNS developmental stages[ Accordingly\ a major dendritic growth between

Fig[ 0[ Basal release of ð2HŁ!GABA from cerebral cortex slices obtained from rats injected with physiological saline solution "CTRL# "*ž*#\ eqNaCl "*T*# or MSG "**# during the _rst 6 postnatal days[ "A# 03 days old^ "B# 10 days old^ "C# 29 days old^ "D# 59 days old[ Data represent the means2SDM from 4 experiments done by duplicate[ Ð  signi_cantly di}erent when compared to CTRL and eqNaCl groups at P ³ 9[990[

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Fig[ 1[ Stimulated ð2HŁ!GABA release from cerebral cortex slices obtained from rats injected with physiological saline solution "CTRL# "*ž*#^ eqNaCl "*T*# or MSG "**# during the _rst 6 postnatal days[ Basal release "**# corresponds to values obtained from CTRL group in each age referred in Fig[ 0[ The stimulus "PULSE# was induced with 42 mM KCl from fractions 3 to 7[ Data represent the mean2SDM from 4 experiments done by duplicate[ "A# 03 days old^ signi_cantly di}erent when compared to CTRL group\ at pP ³ 9[94^ PP ³ 9[994 and when compared to eqNaCl at pP ³ 9[94^ "B# 10 days old^ "P# signi_cantly di}erent at P ³ 9[994 when eqNaCl or MSG were compared to CTRL group\ and when MSG group was compared to eqNaCl^ "C# 29 days old^ "p# signi_cantly di}erent when compared to CTRL and eqNaCl groups at P ³ 9[94^ "D# 59 days old^ "P# signi_cantly di}erent when compared to CTRL and eqNaCl groups at P ³ 9[994[

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Fig[ 2[ ð2HŁ!GABA release over~ow in cerebral cortex slices obtained from rats injected with physiological saline solution "CTRL#^ eqNaCl or MSG during the _rst 6 postnatal days[ The over~ow was calculated by substracting the mean value of the basal release\ to the stimulated ð2HŁ!GABA release "fraction 3 to 7# in the corresponding fraction\ in each group and at the each age studied[ Data represent the absolute average\ and the statistical tests were done considering the fraction of maximal stimulated release from all data shown in Fig[ 1[ "A# 03 days old^ "p# statistically di}erent from CTRL group\ at P ³ 9[94^ and "p# at P ³ 9[994^ "# statistically di}erent when compared to eqNaCl group at P ³ 9[94^ "B# 10 days old^ "# statistically di}erent from eqNaCl group at P ³ 9[994\ and "P# from CTRL group at P ³ 9[994^ "C# 29 days old^ "D# 59 days old^ "P# statistically di}erent from CTRL and eqNaCl groups at P ³ 9[994[

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Fig[ 3[ Basal release of ð2HŁ!GABA from hippocampal slices obtained from rats injected with physiological saline solution "CTRL# "*ž*#^ eqNaCl "*T*# or MSG "**# during the _rst 6 postnatal days[ "A# 03 days old^ "B# 10 days old^ "C# 29 days old^ "D# 59 days old[ Data represent the means2SDM from 4 experiments done by duplicate[ "# signi_cantly di}erent when compared to CTRL and eqNaCl groups at P ³ 9[990[

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Fig[ 4[ Stimulated ð2HŁ!GABA release from hippocampal slices obtained from rats injected with physiological saline solution "CTRL# "*ž*#^ eqNaCl "*T*# or MSG "**# during the _rst 6 postnatal days[ Basal release "**# corresponds to values obtained from CTRL group in each age referred in Fig[ 0[ The stimulus "PULSE# was induced with 42 mM KCl from fractions 3 to 7[ Data represent the mean2SDM from 4 experiments done by duplicate[ "A# 03 days old^ "P# signi_cantly di}erent when compared to CTRL group\ at P ³ 9[994 and when compared to eqNaCl at pP ³ 9[94^ "B# 10 days old^ "P# signi_cantly di}erent at P ³ 9[994 when eqNaCl or MSG were compared to CTRL group\ and when MSG group was compared to eqNaCl^ "C# 29 days old^ "D# 59 days old^ "P# signi_cantly di}erent when compared to CTRL and eqNaCl groups at P ³ 9[994[

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Fig[ 5[ ð2HŁ!GABA release over~ow in hippocampal slices obtained from rats injected with physiological saline solution "CTRL#^ eqNaCl or MSG during the _rst 6 postnatal days[ The over~ow was calculated by subtracting the mean value of the basal release\ to the stimulated ð2HŁ!GABA release "fraction 3 to 7# in the corresponding fraction\ in each group and at the each age studied[ Data represent the absolute average\ and the statistical tests were done considering the fraction of maximal stimulated release from all data shown in Fig[ 1[ "A# 03 days old^ "P# statistically di}erent from CTRL at P ³ 9[994 and "p# from eqNaCl groups at P ³ 9[94 respectively^ "B# 10 days old^ "P# statistically di}erent from eqNaCl group at P ³ 9[994\ and "p# from eqNaCl group at P ³ 9[94^ "C# 29 days old^ "D# 59 days old^ "P# statistically di}erent from CTRL and eqNaCl groups at P ³ 9[994[

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Fig[ 6[ Basal release of ð2HŁ!GABA from striatal slices obtained from rats injected with physiological saline solution "CTRL# "*ž*#^ eqNaCl "*T*# or MSG "**# during the _rst 6 postnatal days[ "A# 03 days old^ "B# 10 days old^ "C# 29 days old^ "D# 59 days old[ Data represent the means2SDM from 4 experiments done by duplicate[

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Fig[ 7[ Stimulated ð2HŁ!GABA release from striatal slices obtained from rats injected with physiological saline solution "CTRL# "*ž*#^ eqNaCl "*T*# or MSG "**# during the _rst 6 postnatal days[ Basal release "**# corresponds to values obtained from CTRL group in each age referred in Fig[ 0[ The stimulus "PULSE# was induced with 42 mM KCl from fractions 3 to 7[ Data represent the mean2SDM from 4 experiments done by duplicate[ "A# 03 days old^ "p# signi_cantly di}erent when compared to CTRL group\ at P ³ 9[94^ PP ³ 9[994 and when compared to eqNaCl at pP ³ 9[94^ "B# 10 days old^ "P# signi_cantly di}erent at P ³ 9[994 when eqNaCl or MSG were compared to CTRL group\ and "p# when MSG group was compared to eqNaCl^ "C# 29 days old^ "P# signi_cantly di}erent when compared to CTRL and eqNaCl groups at P ³ 9[994^ and "p# when compared to CTRL group at P ³ 9[94 only^ "D# 59 days old^ "P# signi_cantly di}erent when compared to CTRL and eqNaCl groups at P ³ 9[994[

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Fig[ 8[ ð2HŁ!GABA release over~ow in striatal slices obtained from rats injected with physiological saline solution "CTRL#^ eqNaCl or MSG during the _rst 6 postnatal days[ The over~ow was calculated by subtracting the mean value of the basal release\ to the stimulated ð2HŁ!GABA release "fraction 3 to 7# in the corresponding fraction\ in each group and at the each age studied[ Data represent the absolute average\ and the statistical tests were done considering the fraction of maximal stimulated release from all data shown in Fig[ 1[ "A# 03 days old^ "p# statistically di}erent from CTRL group\ at P ³ 9[94 and "P# at P ³ 9[994^ "B# 10 days old^ "P# statistically di}erent from CTRL and eqNaCl groups at P ³ 9[994^ and "p# from CTRL group at P ³ 9[94^ "C# 29 days old^ "p# statistically di}erent from CTRL group at P ³ 9[94^ and "p# eqNaCl group at P ³ 9[994^ "D# 59 days old^ "P# statistically di}erent from CTRL and eqNaCl groups at P ³ 9[994[

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days 2 and 10 after birth has been seen in the cerebral cortex associated to the formation of intrinsic GABA! ergic neuronal circuitry and the presence of GABAA receptors between days 02 and 10 after birth "Miller et al[\ 0875"a#\"b#^ Cobas et al[\ 0880^ Marin!Padilla\ 0881#\ in addition to generating inhibitory postsynaptic poten! tials at the end of the _rst week of age "Luhmann and Prince\ 0880^ Avanzini et al[\ 0881#[ Furthermore\ recent experiments suggest that activating NMDA type Glu receptors is linked to neuronal plasticity during the post! natal development[ They are expressed in cortical neu! rones between days 7 and 02 of age before dendritic growth and synapses formation "Lo Turco et al[\ 0880^ Insel et al[\ 0889#[ This suggested an involvement of Glu receptor as mediators of the changes seen in GABA release\ possibly as part of neuronal plasticity[ One should consider\ however\ that Glu receptor activation results in increased intracellular Ca¦¦ concentrations\ not only associated with brain maturation and plasticity with toxicity and neuronal death "Frandsen and Schous! boe\ 0889#[ In this sense\ the possibility of these changes being also the result of GABAergic neuronal loss induced by MSG given at early developmental stages\ cannot be ruled out[ On the other hand\ the results seen in the hippocampus indicate great vulnerability of the GABAergic system to the Glu given at early stages[ These results also suggest an irreversible decrease in neuronal capacity to release GABA\ apparently due to a possible decrease in the num! ber of GABAergic neurons[ Both the baseline release "Fig[ 3# and GABA stimulated release "Figs 4 and 5# are decreased virtually over all ages studied[ This phenom! enon is seen in the hippocampus contrary to what was observed in the cerebral cortex[ It may be accounted for by a balanced interrelationship between the glu! tamatergic and GABAergic systems since the mor! phological substrate in inhibitory events occurs mostly due to GABAergic interneurones "Seress and Ribak\ 0877#[ Little is known\ however\ on the circuitry and transmitters involved in excitatory and inhibitory syn! apses during early life periods "_rst 1 weeks of age#[ There is evidence that hippocampal neurones in immature rats show several electrophysiological characteristics di}erent from those seen in the adult animals such as exogenous GABA release which depolarizes and excites neuronal membranes\ an e}ect mediated by GABAA receptors coupled to Cl− channels[ These GABA!mediated responses appear to be modulated by Glu receptors\ both of the NMDA and the non!NMDA type "Cherubini\ 0882#[ These observations suggest that both systems sup! ply most hippocampus cell excitation and GABA mediated depolarization to also induce increased intra! cellular Ca¦¦\ playing a major role in cell di}erentiation and growth[ During this period there is a synaptic reor! ganization in the hippocampus and a dramatic increase is seen in the receptor expression\ especially in the

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NMDA type of Glu receptors "Wolf and Keilho}\ 0873^ Molino} et al[\ 0883^ Scheetz and Constantine!Paton\ 0883#[ Electrophysiological data on the hippocampus show no neuronal inhibition before day 5 after birth "Harris and Teyler\ 0872#[ Intracellular recordings of cortical and hippocampal neurons con_rm\ however\ the existence of predominantly excitatory synaptic activity in immature animals with major di}erences when compared to activity in the adult rats\ especially responding to GABA as the ability of GABA to depolarize nerve cells in the newborn animal "Cherubini\ 0882#[ Some electrophysiological studies suggest the presence of a glycine regulating site on pyramidal cells making much easier the generation of NMDA mediated giant depolarization potentials in GABAergic neurons\ in addition to GABA mediated and NMDA!regulated potentials "Gaiarsa et al[\ 0889#[ These potentials are inhibited by CNQX while KA increases the frequency of those potentials[ This means that these events are modulated by both receptor subtypes\ NMDA and non!NMDA Glu receptors[ This could\ perhaps\ account for the presence of important signals during syn! aptogenesis as small extracellular Glu quantities promote growth and neuronal di}erentiation in cell cultures "Aru}o et al[\ 0876#[ Although the functional signi_cance of the GABA depolarizing action and the high sensitivity of GABAergic interneurons to Glu agonists is still unknown\ in vitro studies indicate that intracellular Ca¦¦ is essential for neuronal growth and di}erentiation[ Thus\ if GABA increases the intracellular Ca¦¦ concentration\ it also plays a major role in synaptic remodelling during early life stages[ Its functional signi_cance in neuronal excitability might be important[ It has been shown that an increase in intracellular Ca¦¦ due to intensive NMDA receptor stimulation inhibits the development of the growth cones\ while non!NMDA!receptor stimulation seems to mediate dendritic growth retraction "Brewer and Cotman\ 0878#[ A balance between excitation and inhibition allows for controlled neurite extension and the establishment of connections between neurons[ Hence\ in the absence of stimulation\ the growth of cones is slow while with moderate stimulation\ the result is an expan! sion of synaptic connections\ while with high stimulation "hyperexcitation#\ as seen in convulsive crises\ there is a regression\ trimming and elimination of growth cones\ synaptic contacts\ and neuronal death "Sprea_co and Frassoni\ 0882^ Wasterlain and Sankar\ 0882^ Cherubini\ 0882#[ On the other hand\ results from the striatum show that the GABAergic system is vulnerable to neonatal Glu administration[ It induces a reduction of stimulated GABA release at all ages studied "Figs 7 and 8#[ This decrease\ however\ seems to occur only in the GABA releasing capacity by the nerve cells\ as the GABA base! line release is not modi_ed in any age studied "Fig[ 6# suggesting that the number of GABAergic neurons does

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not seem to be modi_ed[ This response from the striatum is di}erent from responses seen in the hippocampus and cerebral cortex[ This could be explained\ in part\ by di}erences in the ontogenetic development between those regions\ i[e[ while the cerebral cortex and the hippo! campus show highly dynamic axonal growth\ dendritic arborization\ synaptogenesis\ synaptic involution\ axonal and cell degeneration\ events are also occurring during the _rst 29 days after birth as signi_cant CNS postnatal developmental stages in rats "Vincent et al[\ 0884^ Oppenheim\ 0880#[ This is not the case with the striatum\ as this region in rodents is morphologically and physio! logically de_ned during the second to third weeks of age "Scheetz and Constantine!Paton\ 0883#[ There are\ however\ two ways to explain these changes of GABA! ergic neurotransmission seen in the striatum\ namely\ "a# a direct overactivation of Glu receptors\ particularly the NMDA type\ located in GABAergic neurons a}ecting GABA!mediated remodelling and neurotransmission at a synaptic level\ as part of the striatal plasticity^ and "b# an indirect e}ect of Glu by an overactivation of NMDA receptors located in other non GABAergic neuronal structures with a major in~uence and e}ects on neuronal remodelling and GABA!mediated neurotransmission as in the case of the dopaminergic system\ among others[ Accordingly\ it has been shown that the GluR0 and GluR1 non!NMDA type of receptor expression is higher in the striatum on day 3 after birth "Pellegrini!Giampietro et al[\ 0880#[ NMDA "Insel et al[\ 0889# and Kainate "Miller et al[\ 0889# type Glu receptors are present since day 0 after birth and are permanent in adult rats while the AMPA type receptors increase to levels close to those in adult rats from day 6 after birth "McDonald et al[\ 0877^ Insel et al[\ 0889#[ These changes could also mean a decrease in the pyridoxal phosphate dependent glutamic acid decarboxylase expression "GAD54# which occurs during the second week after birth "Greif et al[\ 0881# allowing for a lower availability of GABA at the synaptic level[ It would\ therefore\ account for the decreased GABA!stimulated release as seen in Figs[ 7 and 8[ Regarding the second possibility\ there is neuro! chemical evidence that early maturation of other neuro! transmission systems occurs in the striatum and their in~uence on the GABAergic system[ For example\ the cholinergic system maturing during the _rst 2 weeks after birth "Thal et al[\ 0881^ Happe and Murrin\ 0881#^ the dopaminergic system with its D0 and D1 type dopamine "DA# receptors and coupling to second messengers begin! ning day 06 of embryonic age and completing maturation on day 03 after birth "De!Vries et al[\ 0881^ Williams et al[\ 0881#^ and the DA vesicular transport maturing as early as day 7 after birth "Leroux!Nicollet et al[\ 0889#[ Other indicators of early maturation of striatum are the presence of Ca¦¦ binding proteins involved in regulating intracellular Ca¦¦ since day 19 of embryonic age and their consolidation on day 04 after birth "Cimino et al[\

0889\ 0880^ Liu and Graybiel\ 0881#[ And the presence of synaptophysine and synaptoporine over the same period "Ovtscharo} et al[\ 0882#[ Thus\ all of these parameters may correspond to a degree of prenatal maturity with maximal rate of neuroblast mitoses "Zahalka et al[\ 0882#[ GABAergic neurotransmission in this region of the striatum may be indirectly in~uenced for adequate development and integrity of these neurons due to modi! _cation and:or remodelling of other neurotransmitter systems[ For example\ DA seems to control the expression of certain genes such as c!fos\ involved in neuronal transduction during development in critical per! iods such as the _rst 04 days after birth in the striatum "Arnauld et al[\ 0884#[ Also\ perinatal DAergic dea}er! entation with 5!hydroxydopamine in the striatum has been proven to induce changes in the levels of certain aminoacids and GABA which shows the plastic ability of synaptic reorganization occuring in early developmental stages of this region "Molina!Holgado et al[\ 0882#[ In conclusion\ the results of this study show the vul! nerability of the GABAergic system to early exposure to Glu as a monosodium salt during postnatal development of rats indicating the presence of Glu receptors in GABA! ergic neurons while overstimulation of these receptors induces major changes in GABA!mediated neuro! transmission during critical periods for CNS syn! aptogenesis[ One could not infer from the results of this study whether these changes in GABAergic com! munication involve modifying certain behavioral aspects and:or the development of neuronal neuroexcitatory and neurocytotoxic mechanisms[ Therefore\ additional studies are necessary to elucidate how prone neurons are to degenerate and:or determine behavioral changes occurring during older ages[

Acknowledgements This work was partially supported by CONACyT grant 0305!N8196 and constitute part of C[B[Z[ thesis to obtain Doctoral degree at C[U[C[S[\ Universidad de Guadalajara[ The authors thanks to Ms[ Raquel F[ de Shumski for her assitence in the language review of this manuscript[

References Arnauld\ E[\ Arsaut J[\ Tafani J[A[\ Demotes!Mainard\ J[\ 0884[ Dopa! minergic control of gene transcription during striatal ontogeny] c! fos induction by D0 receptor activation in the developing strioso! mes[ Brain Res[ Mol[ Brain Res[ 29\ 112Ð121[ Aru}o\ C[\ Ferszt\ R[\ Hildebrandt\ A[G[\ Cervos!Navarro\ J[\ 0876[ Low doses of L!monosodium glutamate promote neuronal growth and di}erentiation in vitro[ Dev[ Neurosci[ 8\ 117Ð128[ Avanzini\ G[\ Franceschetti\ S[\ Panzica\ F[\ Buzio\ S[\ 0881[ Age! dependent changes in excitability of rat neocortical neurons studied

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