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

Neurochemistry International 61 (2012) 258–265

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Short- and long-term changes in extracellular glutamate and acetylcholine concentrations in the rat hippocampus following hypoxia S.J. López-Pérez ⇑, A. Morales-Villagrán, J. Ventura-Valenzuela, L. Medina-Ceja Departamento de Biología Celular y Molecular, Centro Universitario de Ciencias Biológicas y Agropecuarias, Universidad de Guadalajara, Jalisco, Mexico Carretera Guadalajara-Nogales km 15.5, predio ‘‘Las Agujas’’, Nextipac, Zapopan, Jalisco, CP 45110, Mexico

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Article history: Received 27 September 2011 Received in revised form 8 February 2012 Accepted 13 March 2012 Available online 21 March 2012 Keywords: Hypoxia Neurotransmitters Hippocampus

a b s t r a c t 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. Ó 2012 Elsevier Ltd. All rights reserved.

1. 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 et al., 2010; Duan et al., 2011). This immediate systemic

⇑ Corresponding author. Tel./fax: +52 33 37 77 11 71. E-mail address: [email protected] (S.J. López-Pérez). 0197-0186/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2012.03.009

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 a-ketoglutarate, an intermediate in

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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-MethylD-aspartate (NMDA), a-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 longterm 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.

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32 ± 5.6%) in the dorsal hippocampus under ether anesthesia (AP: 3.2 mm, L: 2.4 mm, D: 2.0 mm: Liu et al., 2005). The probe was perfused during surgery with Krebs–ringer oxygenated solution (in mM):NaCl (118.0), KCl (4.0), KH2PO4 (1.25), MgSO4 (1.17), CaCl2 (2.2), NaHCO3 (25.0), glucose (10). After surgery, the anesthesia was discontinued and the rats were placed in a convenient-size sealed testing chamber (1 dcm3). The inflow and outflow ends of the microdialysis probe were connected with tubing to a swivel at the top of the chamber, permitting simultaneous perfusion and fraction collection. 2.3. Hypoxia protocol and fraction collection After an equilibration period (1 h), fractions were collected every minute at a flow rate of 2.5 ll/min. Animals were exposed to normoxic conditions for 5 min and then hypoxia (8% O2/92% N2) was induced for 45 min, which was followed by a 25 min post-hypoxia period in normoxic conditions. Additional samples were then collected after a further one and two hours (15 fractions after each). Dialysis samples were stored at 20 °C until Glu or ACh was quantified. Animals from the control group (n = 8) were implanted with microdialysis probes on the same day as the experimental animals and they were placed in the testing chamber for the same period of time, although they were not exposed to hypoxic conditions. Separate groups of animals were used to collect samples for Glu and ACh analysis. An additional group of rats (n = 8) was subjected to hypoxia protocol described above on PD7, without having underwent surgery to insert the probe. This group was used when they reached PD30. 2.4. Surgery at PD30 Upon reaching PD30 the animals previously asphyxiated at PD7 that had not been used before were used in a microdialysis study to determine the long-term effects of perinatal hypoxia on basal and stimulated extracellular concentrations of Glu and ACh. The potassium channel blocker 4-aminopyridine (4-AP) was used to stimulate neurotransmitter release having inserted a microdialysis probe (CMA/7, 7 mm shaft length and 2 mm membrane length, mean recovery 32 ± 5.6%) in the dorsal hippocampus under ether anesthesia at PD30 rats (AP: 3.8 mm; L: 3.2 mm; D: 5.0 mm). After an equilibration period of 3 h, dialysis fractions were collected (one sample per mixture at a flow rate of 2.5 ll/ min) according to the following experimental protocol: 1–20 min, basal conditions (4 mM K+ in Krebs–Ringer solution); 21–25 min, conditions of stimulation (25 mM 4-AP in krebs solution, by reverse microdialysis through the same probe; the sodium chloride concentration was reduced to adjust the osmolarity); 26–60 min, basal conditions (4 mM K+ in Krebs–ringer solution). Separate groups of animals were used to collect fractions for Glu and ACh analysis.

2. Materials and methods 2.5. Histological analysis 2.1. Animals Pregnant Wistar rats (n = 32) were housed in independent cages, with 12  12 light cycle and ad libitum access to food and water. At birth, only males were selected to form litters of eight animals. The pups remained with their mother until PD7. All efforts were made to minimize animal suffering, and to reduce the number of animals used. 2.2. Surgery at PD7 PD7 rats were implanted with a microdialysis probe (CMA/7, 7 mm shaft length and 2mm membrane length, mean recovery

After surgery, all animals were sacrificed by intraperitoneal overdose of sodium pentobarbital and brains were removed and cut in 40 lM slides for histological verification of right microdialysis probe placement in dorsal hippocampus, using cresyl violet dye. 2.6. Determination of extracellular Glu and ACh concentrations To determine the Glu concentration, the total volume of each thawed dialysis sample (2.5 ll) was added to an equal volume of an enzymatic reactor containing glutamate oxidase (GluOx, 0.1 U/ ml:Sigma–Aldrich), horseradish peroxidase (HRP, 5 U/ml:Sigma– Aldrich) and Amplex-Red (100 lM:Invitrogen) in Tris–HCl

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buffer(0.1 M, pH 7.5). The samples (5 ll in total) were mixed and transferred to glass capillary tubes (11.5 mm in length), with each series of fractions being run in parallel with a Glu calibration curve (the principle of this procedure is shown in Fig. 1A). The capillaries tubes were placed in an automated carrousel (Innova-Cielec, S.A. de C.V., Mexico) and after 45 min incubation in the dark, each tube was excited with 560 nm light and the emitted fluorescence registered to calculate the neurotransmitter concentration based on the calibration curve. A similar procedure was used to determine the ACh concentration in the dialysate. In this case the enzymatic reactor used consisted of acetylcholinesterase (AChE, 10 U/ml:Sigma–Aldrich) choline oxidase (COx, 0.5 U/ml:Sigma–Aldrich),horseradish peroxidase (HRP, 5 U/ml: Sigma–Aldrich) and Amplex-Red (Invitrogen, 100 lM) in Tris–HCl buffer (0.25 M, pH 8.0). ACh concentrations were measured as indicated above (see Fig. 1B), although it should be noted that this method does not distinguish between choline and ACh. However, this procedure does provide useful information regarding the turnover of this neurotransmitter during particular physiological conditions. 2.7. Statistical analysis Calibration curves for Glu and ACh were determined in triplicate and the average concentration was calculated for each concentration. Regression analysis curves and the calculation of the neurotransmitter concentration were carried out using GraphPad Prism 5 software. Each point in the resulting graphs represents the mean ± standard deviation for the corresponding neurotransmitter concentration (in percentage with respect to basal average of the control group, n = 5 per experimental group). A Student’s t test was performed when two groups were compared (for example, normoxia vs. hypoxia period or hypoxia vs. control groups), with statistical significance considered at p < 0.05.

Fig. 1. Glu and ACh measurement principle. (A) Glu is converted to a-ketoglutarate, ammonium (NH3) and hydrogen peroxide (H2O2) by glutamate oxidase (GluOx). H2O2 reacts with 10-acetyl-3,7-dihydroxyphenoxazine (Amplex-red reagent) in the presence of horseradish peroxidase (HRP), producing the highly fluorescent derivate, resorufin, that can be quantified at 590 nm. (B) ACh is converted to acetate and choline by acetylcholinesterase (AchE). In turn, choline is converted to betaine and H2O2 by choline oxidase (Cox). H2O2reacts with10-acetyl-3,7-dihydroxyphenoxazine (Amplex-red reagent) in the presence of horseradish peroxidase (HRP), producing the highly fluorescent compound, resorufin, that can be quantified at 590 nm.

3. Results Exposure to hypoxic conditions (8% O2/92% N2) resulted in an increased rate of respiration, immobility and a characteristic head clonus, all of which were observed for up to3 h hours after hypoxia induction. Only rats that exhibited all of these characteristics were included in the study. The enzymatic method for Glu determination was sensitive and produced a linear response in the concentration range used (0.75– 25 lM, r2 = 0.997: Fig. 2, insert). Accordingly, it was deemed to be suitable to measure the extracellular Glu concentrations in the hippocampus. The mean concentration of hippocampal glutamate in PD7 rats was 6.60 ± 1.36 lM. A significant increase in the extracellular Glu concentration several minutes after hypoxia was induced, that was maintained throughout the period of hypoxia (6.60 ± 1.36 lM vs. 58.12 ± 14.75 lM, 490%, in basal and hypoxic conditions, respectively; ⁄p < 0.05). Elevated Glu levels persisted for 150 min after terminating the exposure to hypoxia (24.19 ± 17.17 lM, 328%) and notably, we observed large increases in the Glu concentration even between 210–225 min after hypoxia induction (41.57 ± 13.32 lM, 482%, Fig. 2). In this last period (210– 225 min after hypoxia) the trend toward higher Glu concentrations was maintained in the hypoxia group. Interestingly, the basal extracellular Glu levels in PD30 rats exposed to hypoxia at PD7 were significantly lower than those of control animals (2.39 ± 0.24 lM vs. 6.32 ± 0.48 lM, respectively; ⁄p < 0.05, Fig. 3, inset). The administration of 4-AP induced significant increases in glutamate levels in both control (6.32 ± 0.40 vs. 22.52 ± 10.71 lM, 385%, in basal and stimulated conditions, respectively; 1p < 0.05) and hypoxic rats (2.50 ± 1.24 vs. 5.39 ± 4.78 lM, 272%, in basal and stimulated conditions, respectively; ;p < 0.05, Fig. 3). However, Glu release was delayed in hypoxic vs. control animals as maximum glutamate concentrations were observed 7 min after 4-AP administration in the control group but14 min after 4-AP administration in the hypoxia group. Furthermore, in the hypoxia group lower extracellular Glu concentrations were detected than in control animals at all times (Fig. 3). The calibration curves for ACh determination using the enzymatic methodology gave r2 values above 0.95 for a concentration range of 0.75–25 lM (Fig. 4, insert). The basal ACh level in PD7 rats was 3.66 ± 1.26 lM, which decreased significantly after exposure to hypoxia and these lower levels persisted for the remainder of the test period (3.66 ± 1.26 vs. 1.38 ± 1.16 lM, 72%, in basal and hypoxic conditions, respectively; ⁄p < 0.05, Fig. 4). A significant overall difference in the extracellular ACh concentrations during the hypoxia period was also observed between control and hypoxic animals (3.71 ± 1.25 vs. 1.38 ± 1.16 lM in control and hypoxic group, respectively; ⁄p < 0.05, Fig. 4). Basal ACh levels in PD30 rats previously exposed to hypoxia at PD7 were significantly higher than those of control animals (5.24 ± 0.25 vs. 2.06 ± 0.25 lM, respectively; ⁄p < 0.05, Fig. 5, insert). Moreover, intra-hippocampal administration of 4-AP to control animals induced a transient increase in ACh levels, reaching a maximum value of 9.01 ± 0.77 lM, 224%, between 12–14 min after 4-AP administration (Fig. 5; 1p < 0.05 vs. basal levels). By contrast, when 4-AP was administered to hypoxic animals there was no significant effect on ACh levels (Fig. 5).

4. Discussion We have quantified the concentration of extracellular Glu and ACh before, during and after PD7 rats were exposed to hypoxia and we investigated the long term effects of hypoxia by measuring the concentrations of these neurotransmitters in PD 30 rats

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Fig. 2. Glu concentration in PD7 rats. The extracellular Glu concentration increased during hypoxia and it remained elevated throughout the experimental procedure. Comparisons between control vs. hypoxic rats (⁄p<0.05) are shown. Inset: calibration curve generated with Glu standards from 0.75 to 25 lM (r2 = 0.997). Similar curves were generated before each Glu determination.

exposed to perinatal hypoxia. Perinatal hypoxia is a common cause of neonatal seizures and encephalopathy, and it can result in permanent brain damage, although the precise mechanisms underlying these effects remain unknown. To explore the roles of Glu and ACh in these processes, we used a hypoxia model that mimics the oxygen deprivation experienced by newborns during birth complications, like prolonged labor, respiratory distress of the baby, neonatal aspiration syndromes, pulmonary hemorrhage, chronic respiratory disease, and others (McGuire 2007). In a similar way, animals subjected to low-oxygen conditions within the chamber in this work, experienced a lack of oxygen similar to that experienced when neonatal asphyxia occurs: there is a minor entry of oxygen to body through the respiratory system that affects all the corporal economy, and especially the brain. We did not make additional manipulations in the experimental animals, like carotid ligation, because in a ‘‘natural’’ asphyxia process there are not such manipulations, rather the initial hypoxia triggers a series of events that we are interested in studying. The induction of hypoxia in PD7 rats provoked a rapid and dramatic increase in extracellular Glu levels, which was maintained for at least 25 min. Moreover, Glu levels remained elevated with respect to basal levels for approximately two hours after hypoxia. In conjunction with this neurochemical response, head clonus was observed in rats that began several minutes after the induction of hypoxia and that lasted for the duration of the experiment. In the model used here, seizures have been reported in immature rats (Jensen et al., 1991) and indeed, minimal clonic seizures restricted to forelimb and head muscles were described in rats less than one week old in response to procedures that produce formal epileptic seizures in adult rats (Mares et al., 2004; López-Pérez et al., 2010). Given the increase in extracellular Glu observed in PD7 rats, it is likely that this head

nodding is an early manifestation of the hyperexcitability of the immature circuits in response to the increase in Glu levels. There is considerable evidence implicating Glu in hypoxia-induced brain damage. Short periods of hypoxia (15–20 min) increase hippocampal CA1 excitability in rat pups (Jensen et al., 1988), an effect that can be blocked in both neonatal and adult rodents by NBQX, an antagonist of the AMPA receptor (Jensen et al., 1995; Rubaj et al., 2003). Similarly, MK-801 and [3H]-glutamate binding to NMDA receptors decreases in hippocampal slices from neonatal rats exposed to hypoxia, suggesting altered glutamatergic neurotransmission in this brain region (Otoya et al., 1997; Fritz et al., 1996). Our results describe an early and sustained increase in Glu release in the hippocampus, which may be induced by the rapid depolarization of hippocampal neurons observed in response to hypoxia (Lipton and Whittingham, 1979; Ye et al., 2010). Hypoxia impairs astrocyte Glu transport in vitro (Dallas et al., 2007), which may also contribute to the increase in extracellular Glu levels. In the same sense, Jensen and Cols. (Jensen et al. (1998)) shows an important postsynaptic response consisting in a significant increase in the amplitude and duration of excitatory postsynaptic potentials in hippocampal slices from hypoxic pups when compared with controls, lasting for up to 80 min after tetanic stimulation. Taken together, these studies make it reasonable to consider that the time of hypoxia used in this work (45 min) was sufficient to induce an initial response in hippocampal neurons (that would be the first neurons in release Glu) and a secondary response consisting in the postsynaptic activation observed by Jensen and cols. All these observations can represent alterations in the intrinsic excitability of the hippocampus and alterations in excitatory glutamatergic neurotransmission in this region. In another hand, previous work demonstrated that exposure of hippocampal neuron

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Fig. 3. Glu concentration in PD30 rats. Insert: the basal Glu concentration in hypoxic rats was lower than that of controls (⁄p < 0.05). Principal: Stimulated release of Glu was lower in hypoxic versus control rats (⁄p < 0.05). Glu concentration was higher after 4-AP application compared with basal period in both control and hypoxia group (1p < 0.05 and ;p < 0.05, respectively), although in hypoxia group such effect was only transitory.

Fig. 4. ACh concentration in PD7 rats. The extracellular ACh concentration was lower during and after the period of hypoxia compared with control rats (⁄p < 0.05). Inset: calibration curve generated with Ach standards of 0.75–25 lM (r2 = 0.983). Similar curves were generated before each ACh determination.

cultures to Glu (10 lM) for 3 min augments intracellular Ca2+ and excitotoxic cell death (Hilton et al., 2006), suggesting that the

duration of the increase in extracellular Glu we observed was sufficient to produce excitotoxicity.

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Fig. 5. ACh concentration in PD30 rats. Insert: the basal levels of extracellular Ach were higher in hypoxic than in controls rats (⁄p < 0.05). Principal: as expected, 4-AP administration induced a transient increase in ACh release in the control group, although this response was absent in hypoxic rats (⁄p < 0.05compare the hypoxic group and the control group before and after 4-AP administration).

In PD30 rats exposed to hypoxia at PD7there was a decrease in basal Glu concentrations levels when compared with the corresponding controls. These results are not completely in concordance with the hyperexcitability long term after hypoxia described in previous reports (Jensen et al., 1995). In the first report, they used Long Evans rats, aged PD 10–12, with a hypoxia period of only 15– 20 min. All these factors results in a higher susceptibility to present tonic-clonic seizures during the hypoxia induction, and before, when animals become adults, they have also an increased susceptibility to kindling-, PTZ- or fluorothyl- evoked seizures. In this and others works, Jensen and cols, have reported seizure induction only in this specific age window (10–12 PD), but not in older or younger rats. In the present report, animals are Wistar rats, from 7 DP, and the hypoxia period used for us was 45 min. Given these characteristic, hypoxia under our conditions is not considered an epilepsy model. In the same order of ideas, it is possible that the difference in the kind of animals and hypoxia time generates a different effect in the long-term brain excitability, so it would require additional experiments to explore this point. Why we did not find higher levels of Glu in the asphyxiated 30 days old rats is an interesting issue, given the lack of data in this age. In Jensen et al. (1995), the hyperexcitability in adults animals (70–80 days old) previously asphyxiated is defined only in electrophysiological terms, and she did not present biochemical data. Actually, there are few works reporting long-time Glu levels in the brain as a consequence of perinatal asphyxia, and our work is the first study made with a minute-byminute microdialysis methodology. In a previous paper, Kohlhauser et al. (1999) measured Glu levels three months following perinatal asphyxia in rats (90 days old), and they found the major Glu hippocampal content with 5 min of asphyxia, and lowest with 10, 15 and 20 asphyxia minutes, suggesting that Glu hippocampal content decrease as the time of asphyxia increase, just like the results presented here. In these both studies, animals were older than ours, so it would be necessary to make new microdialysis measures in older animals under our conditions (Wistar rats, 45 min asphyxia) in order to make suitable comparisons.

Another point of discussion given from studies made of During et al. (1993) and Luna-Munguia et al. (2011); both works found increases in hippocampal Glu during and immediately after seizures in human patients, in 3 min microdialysis fractions (During et al., 1993) and during evoked-seizures in kindled-rats, in 30 min microdyalisis fractions (Luna-Munguia et al., 2011). Both works took brains with an epileptic history (‘‘natural’’ epilepsy in the case of During, and experimental epilepsy in the case of Luna-Munguia). In our case, animals never developed a total epileptic behavior, only a head nodding starting 3–6 min after hypoxia, and lasting during the experimental manipulation. No further seizures-like behavior was observed in the animals in the period from 7 to 30 days used in this work. So, it is possible that the long-term Glu extracellular (and maybe others neurotransmitters) levels evolve in a different way from those observed in During et al., 1999, and Luna-Munguia et al., 2011. As a possible explanation, it is known that kynurenic acid, an endogenous NMDA antagonist that can work as a seizures inhibitor throughout regulating the Glu release (Konradsson-Geuken et al., 2010; Potter et al., 2010). It was showed that kynurenic acid increased importantly in response to hypoxia (Baran et al., 2001; Ceresoli-Borroni and Schwarcz, 2001), making it possible that kynurenic acid mediates a long-term regulation of Glu release in the hypoxia rats. However, additional experiments are necessary to probe such hypothesis. There are additional causes that could explain the low level of Glu in the PD30 rats, which may be explained by two hypotheses. Increases in the expression of the neuronal Glu transporter EAAC-1 were described in the frontal cortex of 3 month old rats exposed to neonatal hypoxia (Kohlhauser et al., 1999), which could feasibly produce a decrease in basal Glu levels. Alternatively, Glu may be synthesised by neuronal uptake of glutamine released from astrocytes, which is then converted to Glu. Decreased incorporation of glucose into amino acid neurotransmitters, including Glu, has been described during hypoxia, resulting in decreased metabolism of these elements in the brain (Gibson et al., 1981) and decreased glutamine synthetase activity in cortical regions 5 days after hypoxia

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(Swamy et al., 2010). Furthermore, a recent study described a reduction in the rate of NADH oxidation under hypoxic conditions (Galeffi et al., 2011), suggesting that a decrease in oxygen levels can disturb oxidative metabolism in the electron transport chain and limit the rate of ATP production. Such alterations would affect the synthesis of Glu (and possibly other neurotransmitters dependent on glucose metabolism) via the Krebs cycle. The present findings are supported by the recent demonstration that the total hippocampal Glu content in 3-month old rats is significantly reduced after hypoxia–ischemia (Macri et al., 2010). However, further studies are necessary to fully understand the developmental consequences of hypoxia on the synthetic pathways of amino acid neurotransmitters. The long term alterations in Glu concentrations described in the present study may also influence Glu receptor function or expression. Decreased MK-801/NMDA receptor binding was observed 40 days after hypoxia in presence of the allosteric modulator of this receptor (Otoya et al., 1997), although no differences were produced in the density of NMDA receptors in the hypoxic rats. Studies using a hypoxia-induced rat seizure model reported an increase in the amplitude of AMPA-mediated excitatory postsynaptic currents and increased phosphorylation of GluR1 subunits 24 h after hypoxia (Rakhade et al., 2008), as well as reductions in GluR2 expression 96 h after hypoxia (Sanchez et al., 2001). These findings indicate that hypoxia induced changes in Glu neurotransmission probably include alterations in receptor function and/or expression. These alterations should be considered in such analyzes to provide a more complete understanding of the complex regulatory mechanisms that are affected by early hypoxia (reviewed in Jensen, 2002). Our results revealed a decrease in extracellular ACh levels during and immediately after hypoxia induction. Similar findings have been reported in other animal models that involve a certain degree of hypoxia. Indeed, ACh synthesis (measured with [U-14C]-glucose and [2H4]-choline) decreased in the rodent hippocampus and in other brain regions during hypoxia (Gibson and Duffy, 1981; Shimada,1981), while decreased levels of striatal ACh were seen in adult rats exposed to hypoxia (Chleide and Ishikawa, 1990). Furthermore, h electrographic activity, which is related with activation of cholinergic neurotransmission, is reduced in the hippocampus during and after the induction of hypoxia in rats (Sun et al., 2002). In conjunction with our data, these findings demonstrate the high sensitivity of cholinergic system during the first phase of hypoxia, both in the hippocampus and in the other brain regions studied. Moreover, this response appears to occur in both adult and neonatal rodents. When rats subjected to perinatal hypoxia reached 30 days of age, the extracellular levels of hippocampal ACh were greater than those of corresponding control animals. The hippocampus does not produce ACh but rather, it contains cholinergic terminals that originate primarily in the medial septal area-diagonal band complex of the basal forebrain nuclei (Woolf et al., 1984). Adult rats exposed to neonatal hypoxia exhibit a loss of ACh markers in many brain regions in several experimental models of hypoxia (Burke and Karanas, 1990; Kohlhauser et al., 1999; Chathu et al., 2008). The recovery of ACh levels observed in PD30 rats exposed to perinatal hypoxia is likely to be the combined result of neuronal loss in basal forebrain regions and subsequent local activation of the synthetic cholinergic mechanism, as a compensatory response to maintain cognitive function. In support of this hypothesis, decreases in the expression of choline acetyltransferase in the basal forebrain after intermittent hypoxia have already been reported (Row et al., 2007). Moreover, administration of physostigmine, an acetylcholinesterase inhibitor, improves the performance of memory tasks by mice exposed to hypoxia (Bekker et al., 2007). These observations underline the importance of cholinergic activity in this

region, and support our observation of elevated hippocampal ACh concentration at PD30. Notably, this higher level in basal ACh concentration in the hippocampus was accompanied by a lack of response to 4-aminopiridine, a non-specific activator of neurotransmitter release. Whether this effect is due to impaired function or expression of potassium channels, the primary target of 4-AP, or whether it represents a generalized response to any stimulus remains to be established. In conclusion, our results provide the first direct evidence of early initial and long-term alterations in hippocampal Glu and ACh concentrations in rats exposed to perinatal hypoxia. Basal extracellular Glu concentrations increased dramatically during and immediately after hypoxia, although at PD30 they had decreased when compared with controls. By contrast, the decrease in ACh levels observed during hypoxia was no longer evident at PD30 and indeed, the basal levels in hypoxic animals were greater than those of controls. Further research is required to fully understand the mechanisms underlying the effects of hypoxia on neurotransmitter release during postnatal development. Indeed, such knowledge could ultimately lead to the development of early therapeutic interventions for newborns exposed to hypoxia, which could be applied prior to the onset of learning disabilities, hyperactivity or seizures.

Acknowledgements This study was supported by PROMEP (EXB-086) and CONACyTCB (105807)

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