Changes in NMDA-receptor gene expression are associated with neurotoxicity induced neonatally by glutamate in the rat brain

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Abstract

The N-methyl-d-aspartate receptor (NMDA-R) is fully functional in the rat early in embryogenesis, and diverse neuronal plasticity events are regulated through its activation later in postnatal development. On the other hand, systemic administration of glutamate (Glu) to rats at birth induces neuronal degeneration in glutamatergic central nervous system regions via Glu receptor activation. However, it is not known whether an increase in neonatal Glu levels modifies the gene expression of NMDA-R subunits, or if these putative changes are related to gamma-aminobutyric acid-mediated (GABAergic) neurotransmission. We measured, by means of semi-quantitative reverse transcriptase polymerase chain reaction, changes in gene expression of the NMDA-R subunits: NMDA-R1, NMDA-R 2A and NMDA-R 2B in cerebral cortex (CC), striatum (ST) and hippocampus (HP) in the brains of rats treated neonatally with monosodium l-glutamate (MSG). These studies were supported by histological and quantitative analysis of the glia. Our results showed histological evidence of neuronal damage, and increased glial cell number and activity were detected. This was seen mainly in the ST and HP of MSG-treated animals. Significant increases in NMDA-R1, 2A and 2B subunits gene expression was also observed in ST and HP but not in CC, where only NMDA-R 2B was increased in MSG-treated rats. Our data suggest that increases in Glu levels and activation of Glu-receptors after neonatal administration of MSG induce an increase in glial cell reactivity and important changes in NMDA-R molecular composition, with signs of neuronal damage.

Introduction

Glutamate (Glu) is a major neurotransmitter of the central nervous system in mammals. Its excitatory effect is mediated through either ionotrophic receptors such as N-methyl-d-aspartate (NMDA), non-NMDA (AMPA and kainate), or metabotropic receptors (Michaelis, 1998).

Recent findings suggest that Glu receptors (Glu-R), specifically the NMDA receptor (NMDA-R), play a role during CNS development. They mediate functions such as neuron survival, axon and dendritic structure, synaptogenesis and synaptic plasticity (Kleinschmidt et al., 1987, Cotman and Monaghan, 1988, McDonald and Johnston, 1990, Lo Turco et al., 1991, Komuro and Rakic, 1993). Glu-R are also implicated in the remodelling of neuronal shunts related to learning and memory processes (Scheetz and Constantine-Paton, 1994). Taken together, these results indicate the presence of Glu-R in the CNS early in rat embryogenesis. Moreover, Glu-R undergo changes in molecular composition during rat postnatal development (McDonald et al., 1990, Pellegrini-Giampietro et al., 1991, Monyer et al., 1992, Bahn et al., 1994, Michaelis, 1998). Thus, it has been suggested that these molecular events might determine regional and temporal neuron susceptibility to the effects of Glu or its analogues (Sucher et al., 1995, Conn and Pin, 1997).

Recently, molecular studies have deciphered the heteromeric composition of NMDA-R. The receptor is composed of several subunits, termed NMDA-R1 and NMDA2. The latter includes several subtypes ranging from NMDA2A to NMDA2D. The genes coding for each of these subunits have been cloned and sequenced (Kutsuwada et al., 1992, Masu et al., 1993, Molinoff et al., 1994, Sucher et al., 1995, Michaelis, 1998). Moreover, electrophysiological analyses have shown that specific combinations of these subunits, when assembling the receptor molecule, determine neuron sensitivity for Ca++ flux (Monaghan et al., 1989, Monyer et al., 1992). It follows that Glu-R up-regulation during the first days after birth might modify NMDA-R molecular composition, alter interneuronal communication, and consequently obliterate critical processes of normal development in the rat brain (Xia et al., 1995, Zhong et al., 1996).

On the other hand, others and we have shown that systemic monosodium l-glutamate (MSG) administration (4 mg/g body weight given subcutaneously) induces excitotoxic damage in newborn rat brains (first 10 days of postnatal age) (Olney, 1978, Ortuño-Sahagún et al., 1997, Beas-Zárate et al., 1998). This experimental in vivo model could be useful to study the molecular and cellular mechanisms involved in neuronal damage induced by an increase in glutamatergic activity resulting from Glu-R over-activation. Examples of such damage would be hypoxia, perinatal ischaemia, hypoglycaemic shock, epilepsy and neurodegenerative disorders such as Alzheimer disease (Cline et al., 1987, Wasterlain and Sankar, 1993, Romano et al., 1995, Ankarcrona et al., 1995, Portera-Cailliau et al., 1997, Rodriguez et al., 2000). Our previous results, using rats treated with MSG, demonstrated significant changes in choline acetyl-transferase activity, muscarinic receptor binding and GABA release in the cerebral cortex, hippocampus and striatum of rat neonates after Glu-administration (Ortuño-Sahagún et al., 1997, Beas-Zárate et al., 1998). However, these data did not shed light on the nature of these changes. We did not determine whether these changes resulted from modifications of NMDA-R molecular composition or just reflected higher efficiency of GABAergic neurotransmission related to the neuronal plasticity induced by exposure to high Glu concentrations at an early age. Therefore, we reasoned that, by analysing mRNA expression in the cerebral prefrontal cortex, hippocampus and striatum, and combining this with a study of histological parameters, we could shed light on the mechanism by which early excessive NMDA-R expression could induce neuronal degeneration. The aim of the present study was, therefore, to evaluate putative changes in NMDA-R subunits gene expression associated with neurotoxicity in brain regions of rats neonatally treated with MSG. Histological and quantitative data regarding glial cells indicated that profound neuroexcitotoxic changes were correlated with an up-regulation of specific NMDA-R genes.

Section snippets

Materials

MSG and all other chemicals used were purchased from Sigma Chemical Co., St. Louis, MO, Trizol, Taq polymerase and reverse transcriptase (RT) were from GIBCO BRL, MD.

Animals

Pregnant Wistar rats were kept in optimal environmental conditions with free access to water and food, under 12–12 h light–dark cycles, at temperatures ranging between 23 and 25°C, and in separate cages. On the day of giving birth, all litters were adjusted to eight offspring per female. The offspring were administered with 4 mg/g

Morphological findings

When the histological sections of cerebral cortex, striatum and hippocampus were obtained from control animals, normal microscopic structure was observed (Fig. 1A, C and E).

The cerebral cortex from MSG treated animals, studied at 60 days of age, showed disorganization of the cortical layers, with hyperchromatic shrunken nerve cells and widening of the Virchow–Robin space (Fig. 1B).

Although the nerve cells in the striatal region of the MSG-treated animals did not show evident alterations, the

Discussion

Several studies have demonstrated during initial stages of rat developmental, that CNS is highly susceptible to Glu-induced toxicity (Olney, 1978). This damage has been associated and extrapolated to several neurodegenerative disorders and takes place particularly in glutamatergic regions like cerebral cortex, striatum and hippocampus (McDonald and Johnston, 1990).

Diverse stimuli on the CNS, such as neurotoxic chemical substances, neurotoxins from bacterial or viral origin, or simply excessive

Acknowledgements

This work was partially supported by CONACyT grant number 30901-M to C. Beas-Zárate.

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