Research report
Hippocampal nitric oxide synthase in the fetal guinea pig: effects of chronic prenatal ethanol exposure

https://doi.org/10.1016/S0165-3806(97)00184-3Get rights and content

Abstract

The effects of chronic maternal administration of ethanol on nitric oxide synthase (NOS) activity and the numbers of NOS containing neurons, and CA1 and CA3 pyramidal neurons in the hippocampus of the near term fetal guinea pig at gestational day (GD) 62 were investigated. Pregnant guinea pigs received oral administration of 4 g ethanol/kg maternal body weight (n=5), isocaloric sucrose/pair feeding (n=5) or water (n=5), or no treatment (NT; n=5) from GD 2 to GD 61. NOS activity in the 25,000×g supernatant of hippocampal homogenate was determined using a radiometric assay. The numbers of NOS containing neurons, and CA1 and CA3 pyramidal neurons were determined using NADPH diaphorase histochemistry and cresyl violet staining, respectively. The chronic ethanol regimen produced a maternal blood ethanol concentration of 193±13 mg/dl at 1 h after the second divided dose on GD 57. Chronic ethanol exposure produced fetal body, brain, and hippocampal growth restriction and decreased fetal hippocampal NOS activity compared with the isocaloric sucrose/pair feeding, water, and NT experimental groups, but did not affect the number of NOS containing and CA1 or CA3 pyramidal neurons. These data demonstrate that, in the near term fetus, chronic maternal administration of ethanol suppresses hippocampal NOS activity and consequent formation of NO, without loss of NOS containing neurons and prior to loss of CA1 pyramidal neurons that occurs in the adult.

Introduction

Excessive maternal consumption of ethanol during pregnancy can produce teratogenic effects, which can manifest in the human as the fetal alcohol syndrome (FAS) [17]. Ethanol's teratogenic effects in the central nervous system (CNS) are probably the most debilitating manifestation of the FAS and can present as intellectual, behavioral, and/or motor dysfunction 5, 6, 17, 23, 46, 48. Experimental animal and human epidemiologic studies have demonstrated that prenatal ethanol exposure can produce microencephaly 42, 46and that ethanol's teratogenic effects are concentration dependent [9]. One of the CNS target sites of ethanol teratogenesis is the hippocampus, a cerebral cortical structure that is involved in learning and memory 1, 5, 40. Chronic prenatal exposure to ethanol restricts hippocampal growth in the near term fetus [32], and decreases the number of CA1 pyramidal neurons in the adult [1].

It has been proposed that l-glutamate (Glu), an excitatory amino acid neurotransmitter, is neurotrophic during brain development via its action on N-methyl-d-aspartate (NMDA) receptors during critical stages of gestation [7]. Perturbation of the neurotrophic action of the Glu–NMDA receptor system could delay or disrupt normal brain development, leading to CNS dysmorphology and dysfunction observed during postnatal life [35]. Nitric oxide (NO) and cGMP are signal transduction messengers that can be synthesized downstream following NMDA receptor activation 27, 28, 44. NO formation, catalysed by nitric oxide synthase (NOS), can lead to stimulation of soluble guanylyl cyclase and consequent formation of cGMP from GTP. NO has been shown to play a role in neurotransmitter release from brain slices [31]and synaptosomes [36], as well as to play a role in brain development [13]. Altered concentration of NO or cGMP in the brain may result in abnormal brain development. In this regard, a study conducted in intact cultured rat embryos has demonstrated that decreased or increased NO concentration can disrupt normal embryonic development or can be neurotoxic, respectively [33].

NOS catalyses the five electron dependent oxidation of one of the nitrogen atoms of the guanidine group of l-arginine to form l-citrulline and NO, a free radical, gaseous and diffusible molecule [34]. Of the three NOS isoforms identified to date, NOS I (neuronal NOS) and NOS III (endothelial NOS) are constitutive and Ca2+ dependent. NOS containing neurons can be identified by immunohistochemical localization of NOS I and NOS III isoforms [21]and by NADPH diaphorase histochemistry 29, 43, 47. In the hippocampus, NOS containing neurons comprise about 2% of the total neuronal population [49].

There is appreciable experimental evidence to support the concept that prenatal ethanol exposure alters the normal functioning of the Glu–NMDA receptor–NO signal transduction system in the developing hippocampus 25, 32, 37, 39, 40. Chronic prenatal exposure to ethanol decreases the density of NMDA receptors, but does not alter the affinity of the Glu binding sites of the NMDA receptor, as determined in the hippocampus of the near term fetal guinea pig [3]and the adult offspring 25, 41. Chronic maternal consumption of ethanol also suppresses NOS activity in the hippocampus of the near term fetal guinea pig [32]. In vitro or acute in vivo ethanol exposure does not alter the density or affinity of the NMDA receptor [2]or NOS activity 14, 32in the near term fetal guinea pig hippocampus. Thus, it appears that acute ethanol exposure does not adversely affect the binding of Glu to the NMDA receptor or NOS catalytic activity. However, in vitro or acute in vivo ethanol exposure suppresses K+ stimulated Glu release in the fetal guinea pig hippocampus [39]. Hence, underactivation of the Glu–NMDA receptor–NO system may be responsible for the dysmorphology of the CA1 region of the hippocampus, observed in postnatal life, that is produced by chronic prenatal exposure to ethanol 1, 8or by ethanol exposure during the brain growth spurt [10].

The objective of the present study was to determine whether chronic maternal administration of ethanol decreases NOS activity and the numbers of NOS containing (NADPH diaphorase positive) neurons, and CA1 and CA3 pyramidal neurons in the hippocampus of the fetus at near term gestation. The guinea pig was selected as the experimental animal for study due to its extensive prenatal brain development [22]. The chronic regimen of 4 g ethanol/kg maternal body weight/day was chosen because it has been shown to produce microencephaly in the near term fetus [3]including the hippocampus [32], hyperactive behavior that persists into adulthood 1, 15, and a 25% decrease in the number of CA1 pyramidal neurons in adult offspring [1], with minimal maternal, embryonic or fetal lethality.

Section snippets

Chemicals and solutions

Dowex® 50W/50X8-400, N-[2-hydroxyethyl]-piperazine-N′-[2-ethanesulfonic acid] (HEPES), ethylenediaminetetraacetic acid (EDTA) disodium salt, dithiothreitol (DTT), leupeptin, nitroblue tetrazolium, dimethyl sulfoxide, Trizma® Base (Tris) and cresyl violet were obtained from Sigma Chemical (St. Louis, MO). l-[14C]Arginine (331.2 mCi/mmol, 99% radiochemical purity) was supplied by Dupont-New England Nuclear (Lachine, QC). Scintiverse® scintillation fluid and paraformaldehyde were obtained from

Maternal blood ethanol concentration

The chronic ethanol regimen produced a maternal blood ethanol concentration of 193.4±12.5 mg/dl (42.0±2.7 mM, n=5) at 1 h after the second divided dose on GD 57. This value is similar to the data of our previous study [32]. There was no measurable ethanol in the blood of the pregnant guinea pigs that received isocaloric sucrose/pair feeding or water treatment.

Pregnancy outcome

There was no maternal lethality for the ethanol (n=5 litters), isocaloric sucrose/pair fed (n=5 litters), water (n=5 litters) or NT (n=5

Discussion

Nitric oxide, a novel neuronal messenger, appears to play a key neurotrophic role 20, 30, 49as part of the Glu–NMDA receptor system that must function optimally for normal brain development [35]. The objective of this study was to determine whether chronic maternal administration of ethanol decreases NOS activity and the number of NOS-containing (NADPH diaphorase positive) neurons, and CA1 and CA3 pyramidal neurons in the hippocampus of the near term fetal guinea pig. The ethanol regimen used

Acknowledgements

The authors wish to thank Dr. James N. Reynolds for his thoughtful input in the preparation and revision of this manuscript. This research was supported by an operating grant (MT-8073) from the Medical Research Council of Canada. K.A. Kimura is the recipient of a Queen's Graduate Fellowship and an Ontario Graduate Scholarship.

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