Short- and long-term changes in extracellular glutamate and acetylcholine concentrations in the rat hippocampus following hypoxia

Abstract

Hypoxia at birth is a major source of brain damage and it is associated with serious neurological sequelae in survivors. Alterations in the extracellular turnover of glutamate (Glu) and acetylcholine (ACh), two neurotransmitters that are essential for normal hippocampal function and learning and memory processes, may contribute to some of the neurological effects of perinatal hypoxia. We set out to determine the immediate and long-lasting effects of hypoxia on the turnover of these neurotransmitters by using microdialysis to measure the extracellular concentration of Glu and ACh in hippocampus, when hypoxia was induced in rats at postnatal day (PD) 7, and again at PD30. In PD7 rats, hypoxia induced an increase in extracellular Glu concentrations that lasted for up to 2.5 h and a decrease in extracellular ACh concentrations over this period. By contrast, perinatal hypoxia attenuated Glu release in asphyxiated rats, inducing a decrease in basal Glu levels when these animals reached PD30. Unlike Glu, the basal ACh levels in these animals were greater than in controls at PD30, although ACh release was stimulated less strongly than in control animals. These results provide the first evidence of the initial and long term consequences of the hypoxia on Glu and ACh turnover in the brain, demonstrating that hypoxia produces significant alterations in hippocampal neurochemistry and physiology.

Introduction

During the postnatal period, hypoxia due to birth complications is a major cause of brain damage (Pasupathy et al., 2009) and this perinatal hypoxia is an important public health issue in many countries. Depending on the severity, hypoxia can lead to perinatal asphyxia syndrome and hypoxic-ischemic encephalopathy and it is associated with a significant number of newborn deaths. Moreover, survivors of brain injury due to oxygen deprivation have a considerable risk of developing neurological complications such as schizophrenia, learning disabilities, seizures, cerebral palsy and mental retardation (Nelson and Grether, 1999, McNeil et al., 2000, Mitchell, 2009).

Hypoxia is produced by a decrease in oxygen availability to the entire organism, resulting in changes in blood flow to the brain, heart and adrenal glandules (Williams et al., 1993, Guerri et al., 2008, Momen et al., 2009). Respiratory and metabolic acidosis also have been described in hypoxic conditions due to the overproduction of lactic acid by interruption of the Krebs cycle and secondary hyponatremia (Na⁺ plasma concentrations below 135 mmol/L: Moritz and Ayus, 2010, Duan et al., 2011). This immediate systemic response is accompanied by a series of alterations in the brain that affect neuronal function and neurotransmitter turnover. Extracellular acidosis may interfere with the normal function of certain K⁺ channels, such as acid-sensitive K⁺-channels of the tandem P-domain K⁺-channel family (TASKs) and neuronal voltage-gated K⁺ (kV) channels (Xiong et al., 2007, Trapp et al., 2008, Ortiz et al., 2009). Alterations in intracellular Na²⁺ homeostasis (Sheldon et al., 2004) and changes in the normal oscillation of mitochondrial NADPH (Mironov and Richter, 2001, Ratan et al., 2007) have also been demonstrated during hypoxia. Together, these observations suggest that hypoxia induces significant alterations in the normal ionic environment in the brain (Jiang et al., 1992, Muller and Somjen, 2000) and that it affects ATP production. Increases in [Ca²⁺]_i and Ca²⁺-dependent neuronal damage have been described in hypoxic conditions (Oka et al., 2003, Pandit and Buckler, 2009), suggesting that Ca²⁺-dependent vesicle fusion, a pre-requisite for neurotransmitter release, may be affected. These observations suggest that hypoxia may induce changes in the extracellular concentration of neurotransmitters in the brain.

Glutamate (Glu) and acetylcholine (ACh) are two excitatory neurotransmitters that participate in the activation of hippocampal neuronal pathways involved in learning and memory. Significantly, the levels of both neurotransmitters are altered in children exposed to hypoxia during the neonatal period (Sun et al., 2002). In neurons, Glu is mainly produced from α-ketoglutarate, an intermediate in the Krebs cycle, and it accumulates in intracellular synaptic vesicles from which it is released in response to Ca²⁺-induced depolarization. The postsynaptic effects of Glu are mediated by ionotropic receptors, named according to their selective agonist: N-Methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and kainate (KA). These receptors are cation channels and their opening is enhanced by Glu binding to specific sites within the receptor structure, resulting in the production of excitatory postsynaptic potentials (Hassel and Digledine, 2006, Rambhadran et al., 2010, Terhag et al., 2010). In addition to the ionotropic receptors, Glu can also activate eight distinct metabotropic receptors (mGlur1 to mGluR8). These receptors share a common structural design of seven transmembrane domains, typical of G protein-coupled receptors. mGluRs are associated with a wide array of diverse cytoplasmic signaling enzymes, including phospholipase C and adenylate cyclase (Hassel and Digledine, 2006, Traynelis et al., 2010). Glu is released in the hippocampus, acting in the trisynaptic circuit between the dentate gyrus and the CA fields.

ACh is the product of the acetylation of choline by acetyl coenzyme A (acetyl-CoA) that is stored in cytoplasmic vesicles, from which it is released in response to an influx of Ca²⁺. ACh is considered a regulatory neurotransmitter in the brain, where it is involved in memory storage and retrieval, long-term potentiation and attention pathways (Terry, 2006, Buchanan et al., 2010, Bailey et al., 2010). ACh exerts its postsynaptic effects through two types of receptors:nicotinic cation-activated receptors and muscarinic G protein–coupled receptors (Taylor and Brown, 2006, Albuquerque et al., 2009). Importantly, the metabolism of both Glu and ACh metabolism is dependent on oxygen supply.

Increases in extracellular Glu concentrations have been described in animal models involving hypoxia, such as hypoxia–ischemia induced by experimental vascular manipulation (Park et al., 2010), or in brain slices (Dos-Anjos et al., 2009) and cell cultures (Lehmann et al., 2009, Sivakumar et al., 2010). Similarly, the availability of ACh in neuronal tissue during hypoxia has been indirectly explored (Chathu et al., 2008). However, few studies have directly assessed the temporal profile of Glu and ACh in a pure in vivo model of hypoxia, uncomplicated by cerebral ischemia. The neonatal seizure model (Jensen et al., 1995) is a useful model to actively monitor the extracellular concentration of neurotransmitters in conscious animals during and after the induction of hypoxia. In the present study, we have investigated the immediate and long-term effects of neonatal oxygen deprivation on extracellular levels of Glu and ACh in the hippocampus of freely moving rats. We observed a large increase in extracellular Glu levels during hypoxia in rats on postnatal day (PD) 7, which lasted for up to 2 h after the induction of hypoxia. Upon reaching PD30, these animals exhibited lower basal Glu concentrations than control animals. Extracellular ACh concentrations decreased in PD7 rats during hypoxia, although they surpassed the control levels by PD30.