Prenatal hypoxia-ischemia alters expression and activity of nitric oxide synthase in the young rat brain and causes learning deficits

Abstract

Inhibition of nitric oxide synthase (NOS) is known to possibly impair learning and memory. Our previous studies have demonstrated that prenatal hypoxia-ischemia (HI) decreases NOS expression and NOS activity in the neonatal rat brain. To investigate whether effects of prenatal HI on NOS expression continue and whether prenatal HI affects learning and memory in young rats, NOS expression and NOS activity were determined in the hippocampus of rat brains at 28 days of age following a prenatal HI insult on G17. Performances in the passive avoidance test and the Morris water maze test were also studied in these young rats prior to sampling. Rat fetuses were subjected to either a 30-min prenatal HI insult or a sham operation (SH) on gestation day 17 and rat pups were delivered naturally. Increased locomotor activity was observed in the prenatal HI rats as compared to the SH rats on postnatal days 13 and 15, but not on postnatal days 20 and 30. Prenatal HI affected learning ability in these young rats at 28 days of age, as indicated by a delayed acquisition of passive avoidance and by longer escape latency in the Morris water maze test as compared to the SH group. Prenatal HI did not affect retention of passive avoidance and spatial memory. Concomitant with these learning deficits, expression of neuronal NOS and endothelial NOS mRNAs as well as Ca²⁺-depedent NOS activity in the hippocampus of the prenatal HI rat brain were significantly decreased as compared to the SH group. These results suggest that a 30-min prenatal HI insult on gestation day 17 in rats has long-lasting effects on NOS expression and NOS activity in the offspring brain and on learning ability of these young rats. The learning deficit in offspring is possibly associated with the reduction in expression of NOS mRNA and NOS activity in the hippocampus of these animals.

Introduction

Nitric oxide (NO) is a neural messenger molecule in the central nervous system. Under pathological conditions such as cerebral hypoxia-ischemia, NO may mediate neuronal death and brain damage 9, 16, 25. In normal physiological processes, NO also serves a wide range of functions, including regulation of cerebral blood flow, neurotransmission, learning and memory [22]. NO is generated from L-arginine by nitric oxide synthase (NOS). There are three isoforms of NOS: calcium-dependent neuronal NOS (nNOS), endothelial NOS (eNOS), and calcium-independent inducible NOS (iNOS) 5, 11. Recent studies have provided substantial evidence that NOS is closely associated with long-term potentiation (LTP), a persistent increase in synaptic activity implicated in certain forms of learning and memory 18, 27, 38. Blockade of NOS activity in the brain with NOS inhibitors has been shown to impair learning and memory in both adult 10, 28, 29, 34, 39 and neonatal rats [24].

In our previous studies, we have found that prenatal hypoxia-ischemia (HI) decreases expression of nNOS in the brain of juvenile rats [7]. Although a 30-min HI on gestation day 17 did not result in apparent brain tissue damage as examined by a conventional histopathological method 6, 8, the insult did significantly reduce NOS activity and expression of nNOS mRNA and protein in the rat brain at postnatal day 14 [7]. Lizasoain et al. [20] have shown that NOS activity in the fetal and developing rat brain reaches a peak at postnatal day 11 and then remains high at the adult level. The peak of NOS activity in the developing rat brain was found preceding the development of synaptic structure and, therefore, is proposed to have an important role in synaptogenesis [20]. It is possible that the prenatal HI-induced reduction in NOS expression and activity at this critical stage may have important consequences on learning ability of the developing rat brain.

Although it varies somewhat with the animal model, the hippocampus has been shown to be the brain area most vulnerable to HI insult in both the gerbil and the neonatal rat model 17, 36. The hippocampus is also a target brain area for the prenatal HI-induced biochemical alterations [32]. Hippocampal lesions have been shown to be closely associated with spatial learning and memory impairment in Morris water maze [23] and with nonspatial learning impairment, such as passive avoidance task [19]. In the current study, we measured NOS activity and expression of NOS isoform mRNAs in the hippocampus of the brain of 4-week-old rats that experienced an intrauterine HI episode at day 17 of gestation. We wanted to see whether effects of prenatal HI on NOS expression and activity in the brain of offspring proceed. A second aim of this study was to examine whether prenatal HI affects learning ability in these young rats. We assessed both spatial and nonspatial learning and memory in these animals, using the Morris water maze and the passive avoidance task.