Physiology & Behavior 123 (2014) 187–192
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Physiology & Behavior journal homepage: www.elsevier.com/locate/phb
Guanfacine ameliorates hypobaric hypoxia induced spatial working memory deficits H. Kauser, S. Sahu, S. Kumar, U. Panjwani ⁎ Defence Institute of Physiology and Allied Sciences (DIPAS), Defence Research and Development Organization (DRDO), Lucknow Road, Timarpur, Delhi 110054, India
H I G H L I G H T S • HH results in PFC dependent cognitive deficits and PFC apoptotic cell death. • GFC administration improves performance of PFC dependent DAT task during the HH. • GFC also protects apoptotic neuronal degeneration in PFC under HH.
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Article history: Received 12 December 2012 Received in revised form 14 May 2013 Accepted 22 October 2013 Keywords: Hypobaric hypoxia Guanfacine Prefrontal cortex Spatial working memory Apoptosis Neurodegeneration
a b s t r a c t Hypobaric hypoxia (HH) observed at high altitude causes mild cognitive impairment specifically affecting attention and working memory. Adrenergic dysregulation and neuronal damage in prefrontal cortex (PFC) has been implicated in hypoxia induced memory deficits. Optimal stimulation of alpha 2A adrenergic receptor in PFC facilitates the spatial working memory (SWM) under the conditions of adrenergic dysregulation. Therefore the present study was designed to test the efficacy of alpha 2A adrenergic agonist, Guanfacine (GFC), to restore HH induced SWM deficits and PFC neuronal damage. The rats were exposed to chronic HH equivalent to 25,000 ft for 7 days in an animal decompression chamber and received daily treatment of GFC at a dose of 1 mg/kg body weight via the intramuscular route during the period of exposure. The cognitive performance was assessed by Delayed Alternation Task (DAT) using T-Maze and PFC neuronal damage was studied by apoptotic and neurodegenerative markers. Percentage of correct choice decreased significantly while perseverative errors showed a significant increase after 7 days HH exposure, GFC significantly ameliorated the SWM deficits and perseveration. There was a marked and significant increase in chromatin condensation, DNA fragmentation, neuronal pyknosis and fluoro Jade positive cells in layer II of the medial PFC in hypoxia exposed group, administration of GFC significantly reduced the magnitude of these changes. Modulation of adrenergic mechanisms by GFC may serve as an effective countermeasure in amelioration of prefrontal deficits and neurodegenerative changes during HH. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Hypobaric hypoxia (HH) encountered during ascent to high altitude is a stressful condition as it is associated with a decreased partial pressure of oxygen leading to reduced oxygen delivery to tissues [1]. Brain in particular is highly vulnerable to such hypoxic stress due to its high oxygen requirement and results in higher order cognitive dysfunctions [2]. The cognitive impairment induced by high altitude exposure draws special concern because these problems compromise a Abbreviations: HH, hypobaric hypoxia; PFC, prefrontal cortex; GFC, Guanfacine; SWM, spatial working memory; DAT, delayed alternation task. ⁎ Corresponding author at: Neurophysiology Division, Defence Institute of Physiology and Allied Sciences (DIPAS), Defence Research and Development Organization (DRDO), Lucknow Road, Timarpur, Delhi-110054, India. Tel.: +91 1123883203; fax: +91 1123914790. E-mail addresses: [email protected], [email protected] (U. Panjwani). 0031-9384/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.physbeh.2013.10.028
person's performance while carrying out highly demanding mental functions requiring attention, vigilance, judgment and working memory. Working memory is thought to lie at the core of many higher order cognitive functions and is defined as the ability to transiently hold information in mind for subsequent manipulation in order to guide goaldirected behavior [3]. The working memory is regulated by prefrontal cortex (PFC) and is created by networks of PFC neurons engaged in recurrent excitation generating persistent activity [4]. The PFC recurrent excitatory firing is profoundly altered by the norepinephrine (NE) and thereby influencing the working memory functions [5]. Chronic exposure to HH is known to cause NE dysregulation [6], working memory deficits [7] and neurodegeneration in brain regions related to learning and memory [8]. This suggests that NE dysregulation under HH may be a possible underlying mechanism leading to cognitive deficits and associated morphological damage. Therefore, targeting NE mechanism in PFC regulating working memory is likely to solve the problems of PFC dysfunctions associated with high altitude ascent.
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Alpha-2 adrenergic agonist is very well known to improve PFC cognitive functions including attention, working memory and behavioral inhibition [9]. Several studies have shown the attenuating influence of alpha-2 adrenergic agonists on the behavioral and morphological consequences in animal model of memory loss [10]. Alpha-2 adrenergic agonist also protects PFC dysfunction and neuronal damage in cerebral ischemia [11]. The neuroprotective effect of alpha-2 adrenergic agonist is exerted by modulation of both presynaptic and postsynaptic mechanisms [12]. Presynaptically, it reduces excitatory neurotransmitter release which is thought to be the underlying cause of hypoxia induced excitotoxicity [13]. Postsynaptic variation includes inhibition of adenylate and guanylate cyclases and thus reduces the neurodegenerative changes induced by excessive intracellular signaling [14]. Moreover, both in vivo and in vitro studies have shown that the beneficial effects of alpha-2 adrenergic agonists are mediated exclusively via the α2A subtype [15]. Although HH induced dysfunction presents a similar picture as seen in other conditions of PFC dysfunction like Schizophrenia [16] and ADHD [17] where Guanfacine, a specific alpha-2A adrenergic agonist proved to be beneficial, its possible beneficial effect in HH has not been examined. Thus, the present study examined the efficacy of Guanfacine (GFC), as a potential therapeutic agent for HH induced PFC cognitive deficit and neuronal damage. 2. Materials and methods
administration. Rats received intra muscular injection of the drug (1 mg/kg body weight), or sterile saline (equal volume) daily during hypoxic exposure for 7 days 15 min before behavioral testing. The 1 mg/kg body weight of the GFC has been reported to be effective to enhance spatial working memory without any sedative, hypotensive or cumulative effects [18,19]. 2.4. Cognitive assessment Rats were trained to learn and perform delayed alternation task (DAT) to test their spatial working memory according to the protocol described by Deacon et al. with some modifications [20]. All animals were maintained on a restricted diet at 80% of their free-feeding weight throughout the experiment. They were initially habituated to a T-maze until they started eating chocolate chips placed in the food wells at the end of each arm. After habituation, training was given until rat's performance stabilized at criterion performance (80% correct choice) on two consecutive sessions (24 h apart). This allowed detection of change in performance with the treatment. At the first trial, rats were given chocolate chips for traversing the runway and entering either arm. Thereafter, for a total of 10 trials per session, they were rewarded only on choosing the maze arm which was not previously visited. Performance was evaluated as the number of trials correct out of 10. Rats were tested 4 times during the HH exposure on testing days i.e. day 1, day 3, day 5 and day 7.
2.1. Animal and experimental protocol 2.5. Tissue processing and microscopy Adult male Sprague–Dawley rats with an average body weight of 240–260 g were used in the present study. Animals were housed in cages (46 cm × 24 cm × 20 cm) with two animals per cage in an air conditioned temperature (22 ± 2 °C) and humidity (55–60%) controlled room with a 12 h light and dark cycles in the animal house of the institute. Rats were provided with food pellet (Lipton India Ltd., India) and water ad libitum until the behavioral study. Experiments to be conducted were approved by the Institutional Animal Ethical Committee. The guidelines documented in the National Institutes of Health's Guide for the Care and Use of Laboratory Animals were followed and all efforts were made to minimize animal suffering. Experimental animals were divided into four groups, Normoxia + Saline (N + SAL, n = 6), Normoxia + Guanfacine (N + GFC, n = 6), Hypoxia + Saline (H + SAL, n = 6) and Hypoxia + Guanfacine (H + GFC, n = 6). On 1st, 3rd, 5th and 7th day of hypoxia exposure, cognitive assessment was done. Guanfacine was administered each day 15 min before cognitive assessment. On the 8th day rats were sacrificed to study their brain morphology.
After HH exposure the animals were perfused transcardially first with ice-cold PBS (0.1 M, pH 7.4) followed by fixation using ice-cold 4% paraformaldehyde (dissolved in 0.1 M PBS, pH 7.4). The brains were dissected out and post-fixed in the same fixative for 24 h at room temperature. The paraformaldehyde-fixed brain were cryoprotected by graded sucrose (10%, 20% and 30%, respectively) solution and 30 μm-thick coronal sections were cut using a cryostat (Leica-3050, Germany) and were placed in 24 well tissue culture plates. Six coronal sections per animal were collected from the PFC at the bregma + 3.20 to + 2.20 mm. The sections were stored at 4 °C until staining. Light microscopy and fluorescence microscopy were performed for visualization. The photomicrographs of the region of interests were captured with a digital camera (Nikon Tokyo Japan) attached to the microscope (Olympus) at ×400 magnification. The neurons were counted from the pyramidal layer II of the medial PFC using ‘Image J’ software (NIMH, U.S.A). The number of neurons was expressed per 55052 mm2 area.
2.2. Exposure to hypobaric hypoxia
2.6. Apoptosis
Exposure to simulated high altitude of 25,000 ft was performed in a specially designed animal decompression chamber (Seven Star, India) operated under a 12 h light–dark cycle which reduced the barometric pressure (atmospheric pressure equivalents to 282 mmHg, PO2 59 mmHg); temperature and relative humidity were maintained at 28–30 °C and 55–60% respectively. The chamber was continuously flushed with fresh air (5.5 l/min) to replenish O2 consumed by rats and remove CO2 produced. The desired altitude (i.e. 25, 000 ft) was attained at an ascent rate of 300 m/min over a period of 20 min. The chamber was brought down to sea level pressure in the morning at 10 o'clock for 1 h every day to replenish food and water, drug treatment and cognitive assessment. Rats were exposed to HH conditions for 7 consecutive days.
2.6.1. Chromatin condensation Hoechst 33342 (Sigma, U.S.A), chromatin dye, freely enters living cells and therefore stains the nuclei of viable cells as well as those undergoing apoptotic or necrotic death. Apoptotic cells were distinguished from viable and necrotic cells by nuclear condensation and fragmentation. Cryostat sections were permeabilized with 0.1% triton in PBS and stained with Hoechst 33342 (10 μg/ml). The stained sections were visualized in UV light and nuclear damage was documented by counting the number of Hoechst positive cells emitting bright fluorescence and exhibiting nuclear morphology.
2.3. Pharmacological intervention Alpha-2A adrenergic agonist, Guanfacine Hydrochloride (Sigma, U.S.A) was dissolved in sterile saline freshly each day before
2.6.2. DNA fragmentation In situ labeling of DNA fragmentation by TUNEL staining was used to study the apoptotic activity. ApopTag Red In Situ Apoptosis Detection Kit (Millipore, U.S.A) was used as per manual instruction for detection of nucleases activated DNA fragments of approximately 200 base pairs. DNA strand breaks were detected by enzymatically labeling the free 3′-OH termini with digoxigenin nucleotides followed by binding
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of fluorescein conjugated anti-digoxigenin antibody. Fluorescent signal was visualized with appropriate excitation/emission filter pairs. The red fluorescence TUNEL positive neurons were counted. 2.7. Neurodegeneration 2.7.1. Neuronal morphology Nissle staining was performed to study the neuronal morphology. The section was immersed in 0.1% cresyl violet (Sigma, U.S.A) for 2 min. Subsequently, it was thoroughly washed and again dehydrated by graded alcohol, cleared, and finally mounted in D.P.X. The small sized, dense, irregularly shaped pyknotic cells were considered as dead and were counted manually in both hemispheres of the medial PFC. 2.7.2. Neuronal degeneration For neurodegeneration study the brain sections were stained with Fluoro Jade-B (Millipore, U.S.A), a poly anionic fluorescence derivative which sensitively and specifically binds to degenerating neurons. The sections were immersed in 1% NaOH in 80% ethanol for 2 min followed by another immersion in 70% ethanol for 2 min and then washed in distilled water for 2 min. The sections were then transferred to fresh 0.06% potassium permanganate solution for 15 min and gently shaken on a rotating platform. This was followed by rinsing in distilled water and transferring to Fluoro Jade B stain where they were gently agitated for 20 min. After staining, the sections were rinsed in distilled water for 2 min, thrice each. Excess water was drained off the slides, and the slides were rapidly dried on a hot plate. The slides were cleared in xylene and then mounted with D.P.X. The sections were visualized using a filter system FITC, and a bright green fluorescence signal indicated degenerating neurons.
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2.8. Statistical analysis All the results were expressed as Mean ± S.E.M. Group differences of behavioral and morphometric data were compared by using oneway ANOVA. Wherever significant differences were found, Bonferroni multiple comparison test was performed as a post hoc analysis to compare the differences between the groups. Difference below or equal the probability level 0.05 was considered statistically significant. 3. Results 3.1. Cognitive assessment 3.1.1. Training Percentage of correct choice was taken as a measure of performance in T maze delayed alternation task (DAT) to test the prefrontal cortical functions. All rats had been trained on this task to an equivalent level of performance. Correct percentage in each group was statistically compared with the baseline value separately in all the groups to illustrate performance over time. With training, correct percentage increased and it is statically significant at last two consecutive sessions (D6, p b 0.001; D7, p b 0.001) of training (Fig. 1A) indicating the learning of DAT during the training period. 3.1.2. Spatial working memory At the end of training, rats were exposed to simulated hypobaric hypoxia equivalent to 25,000 ft for 7 days and were assessed for cognitive ability 4 times over the 7 days. Percentage of correct choice significantly decreased (p b 0.001) after 7 days hypobaric hypoxia exposure when compared to baseline (D0). There is a regular trend in performance impairment in the exposed group during the hypoxia and the change
Fig. 1. Effect of Guanfacine on delayed alteration t-maze task under hypobaric hypoxia. (A) Training of delayed alternation T-Maze task. Percentage of correct choices, an indicator of working memory performance was 80% in the last two consecutive sessions showing learning of the task. (B) Performance of rats during exposure to hypobaric hypoxia for different durations. Percentage of correct choice increased in rats treated with Guanfacine as compared to the hypoxic group in the last two sessions. (C) Working memory performance after 7 days of exposure to hypobaric hypoxia. Performance increased by Guanfacine treatment in rats exposed to 7 days of hypoxia. (D) Perseverance was measured by consecutive errors after 7 days of exposure to hypobaric hypoxia. Consecutive errors increased after hypoxia exposure whereas the errors decreased with Guanfacine treatment during hypoxia. The values represented as Mean ± SEM. *p b 0.05 and ***p b 0.001 versus normoxia and #p b 0.05 and ###p b 0.001 versus hypoxia. Normoxia + Saline (N + SAL), Normoxia + Guanfacine (N + GFC), Hypoxia + Saline (H + SAL) and Hypoxia + Guanfacine (H + GFC).
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was significant at D3 (p b 0.05), D5 (p b 0.01) and D7 (p b 0.001) when compared to D0 (Fig. 1B) indicating consistence deficits on PFC dependent task during the hypoxia exposure. In drug treated group, GFC was administered daily during the hypoxia exposure and in this group performance reached to baseline value (D0) after 7 days of hypobaric hypoxia (Fig. 1C). This amelioration of the PFC deficit by drug administration during the hypoxia exposure is evident at all testing time points i.e. D1, D3, D5 and D7 but it is statistically significant during the two last session (D5, p b 0.5; D7, p b 0.001) of testing (Fig. 1B). Perseveration, defined as the mean number of consecutive errors per test session is another parameter to test PFC functions (Fig. 1D). There was an increase in perseveration in hypoxia exposed rats (p b 0.001) and it was rescued in the hypoxic group treated with the GFC (p b 0.05). These results indicate improvement in the performance of a PFC dependent task by GFC administration during the hypoxia exposure.
3.2. Apoptosis 3.2.1. Chromatin condensation Chronic exposure for 7 days was found to increase chromatin condensation by 11 fold (29.33 ± 1.71 Hoescht Positive Neurons/ 55052 mm2) in medial PFC compared to normoxia group (2.50 ± 0.50, p b 0.001) (Fig. 2A–B). GFC administration decreased chromatin condensation by 3 fold (7.83 ± 0.31) as compared to hypoxia exposed animals (p b 0.001). This indicates the GFC rescue chromatin condensation which is characteristic of cells undergoing apoptosis under HH.
3.2.2. DNA fragmentation A 13 fold (26.33 ± 0.67 TUNEL Positive Neurons/55052 mm2) increase in DNA fragmentation was observed in medial PFC on 7 days of exposure to HH as compared to the normoxic animals (2 ± 0.37, p b 0.001) (Fig. 2C–D). The number of TUNEL positive neurons however decreased by 4 fold (26.33 ± 0.67) on the administration of GFC during HH than hypoxia exposed group (p b 0.001). This result is in validation of the role of GFC under HH to protect neuronal apoptosis in term of DNA fragmentation in the medial PFC. 3.3. Neurodegeneration 3.3.1. Neuronal morphology In case of normoxia group there was no alteration of morphology but exposure to HH showed pyknotic and tangle-like appearance along with large dot like neurons in medial PFC. The pyknotic cell count in medial PFC was increased by 29 fold (26 ± 2.44) after 7 days of hypoxia exposure compared to normoxia group (0.88 ± 0.15, P b 0.001) (Fig. 3A–B). Daily treatment with GFC for hypoxia exposed animals minimized the neuronal damage by 6 fold (4.24 ± 0.44) as compared to animals exposed to HH without treatment (p b 0.001) indicating the neuroprotection of hypoxia induced neurodegeneration in medial PFC by GFC administration. 3.3.2. Neuronal degeneration Exposure to HH for 7 days showed 19 fold increase in Fluoro Jade-B positive neurons (27.33 ± 1.05) in medial PFC as compared to normoxia
Fig. 2. The effect of Guanfacine on hypobaric hypoxia induced apoptosis. (A) Chromatin condensation by Hoechst 33342 staining. (B) Graph showing the change in number of Hoechst positive neurons. (C) DNA fragmentation by TUNEL assay. (D) Graph showing the change in number of TUNEL positive neurons. Normoxia + Saline (N + SAL), Normoxia + Guanfacine (N + GFC), Hypoxia + Saline (H + SAL) and Hypoxia + Guanfacine (H + GFC). Bars represent Mean ± SEM of average of six fields in six individuals of each group. *** denotes p b 0.001 when compared to normoxic group and ### denotes p b 0.001when compared to hypoxic group. The arrows indicate apoptotic neuron (magnification ×400).
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Fig. 3. The effect of guanfacine on hypobaric hypoxia induced Neurodegeneration. (A) Neuronal pyknosis by Nissle staining. (B) Graph showing the change in the number of dead neurons. (C) Neuronal degeneration as is shown by Fluoro Jade B staining. (D) Graph showing the change in number of Fluoro Jade B positive neurons. Normoxia + Saline (N + SAL), Normoxia + Guanfacine (N + GFC), Hypoxia + Saline (H + SAL) and Hypoxia + Guanfacine (H + GFC). Bars represent Mean ± SEM of average of six fields in six individuals of each group. *** denotes p b 0.001 when compared to normoxic group and ### denotes p b 0.001when compared to hypoxic group. The arrows indicate neurodegenerative neuron (magnification ×400).
group (1.42 ± 0.30, p b 0.001) (Fig. 3C–D). The neuronal degeneration in GFC treated hypoxic groups was lowered by 6 fold (4.04 ± 0.20) than animals subjected to similar duration of hypoxia exposure but without drug administration (p b 0.001). This result further validated the neuroprotection of medial PFC neurons by GFC administration during the HH. 4. Discussion The results presented herein demonstrate that exposure to HH impaired spatial working memory performance on the DAT. This is consistent with previous findings showing the working memory impairment under hypoxic condition using different testing paradigms [21]. DAT is a specific test of PFC dependent spatial working memory [22] and working memory has been shown to be highly influenced by NE in PFC [23]. HH induced spatial working memory deficits observed in the present study might also be correlated with dysregulation in NE transmission as in other stressful conditions [24]. Acute stress impairs prefrontal cortical function through a cascade of events: There is a large increase in NE release in the PFC, which in turn leads to high levels of cyclic adenosine monophosphate (cAMP) and protein kinase C (PKC) intracellular signaling, which reduces prefrontal neuronal firing, and rapidly impairs working memory [25]. When stressors are repeated over many days and weeks, there are neurodegenerative changes attributed to enhance intracellular signaling stimulated by altered levels of NE [26]. A systemic administration of GFC (1 mg/kg b.w.), an alpha-2A adrenergic agonist during the hypoxia exposure showed improvement in spatial working
memory and this change was significant after 5 and 7 days of hypoxia exposure when neuronal damage has been reported to be maximum [27]. This indicates that exposure to HH induced a dramatic and progressive impairment of PFC dependent task in vehicle-treated rats, growing more pronounced over the duration of the hypoxia exposure whereas stimulation of alpha-2A receptors with daily GFC treatment rescued DAT performance in exposed rats. The morphological data to study apoptosis and neurodegeneration in medial PFC showed that there was increased cell damage in medial PFC after 7 days of hypoxia exposure. Lesion and pharmacological studies in rodents have implicated the role of medial prefrontal cortex in working memory tasks [28]. Chronic stress severely disrupts two key processes attributed to the PFC: working memory and behavioral flexibility; these behavioral deficits closely correlated with a selective reduction in the volume of the upper prefrontal layers (I and II) [29]. Although hypoxia has been reported to cause spatial working memory impairment, it has not been examined if these deficits could be due to neuronal damage in the PL/IL cortices, structures within the medial prefrontal cortex of rodents that are critical for working memory. The loss of these neurons is expected to have great consequences for cognitive performance. Using a HH animal model, the present study has revealed that the apoptosis and neurodegeneration increased in the PL/IL tissue of exposed animals, which was associated with cognitive deficit. Exposure to HH resulting in a significant reduction in neuronal number may be due to the stress associated with hypoxia [30]. Daily treatment of GFC during the hypoxia exposure rescued the neuronal damage significantly in these brain regions. Interestingly, the behavioral results corroborated
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with the hypoxia induced cortical damage. The beneficial effect of GFC treatment was most pronounced in the late phase of the exposure when morphological changes were observed. These findings reiterate that altered NE transmission contributes to prefrontal dysfunction and neuronal damage, and that stimulation of alpha-2A receptors may provide relief for the working memory deficits observed in HH. 5. Conclusion Modulation of adrenergic mechanisms by GFC may play a role in the amelioration of prefrontal deficits and neurodegenerative changes during hypobaric hypoxia. It appears that the neurodegeneration has a role in cognitive dysfunction and protecting prefrontal regions vulnerable to hypoxia may be a key factor in rescuing prefrontal cortical regulation of behavior and cognition. Therefore we suggest that long lasting PFC dependent cognitive impairment observed during hypobaric hypoxia may be due to neuronal damage in PFC and these devastating changes can be ameliorated by administration of alpha 2A adrenergic agonist. Acknowledgments This study was supported by Defence Research and Development Organization, Ministry of Defence, Government of India and Department of Science and Technology, Government of India. References [1] Savourey G, Launay JC, Besnard Y, Guinet A, Travers S. Normo- and hypobaric hypoxia: are there any physiological differences? Eur J Appl Physiol 2003;89(2):122–6. [2] Lieberman P, Protopapas A, Reed E, Youngs JW, Kanki BG. Cognitive deficits at high altitude. Nature 1994;372:325. [3] Baddeley A. Working memory. Science 1992;255:556–9. [4] Goldman-Rakic PS. Cellular basis of working memory. Neuron 1995;14:477–85. [5] Arnsten AFT. Through the looking glass: differential noradrenergic modulation of prefrontal cortical function. Neural Plast 2000;7:1–2. [6] Saligaut C, Chretien P, Daoust M, Moore N, Boismare F. Dynamic characteristics of dopamine, norepinephrine and serotonin metabolism in axonal endings of the rat hypothalamus and striatum during hypoxia: a study using HPLC with electrochemical detection. Methods Find Exp Clin Pharmacol 1986;8(6):343–9. [7] Kramer AF, Coyne JT, Strayer DL. Cognitive function at high altitude. Hum Factors 1993;35(2):329–44. [8] Maiti P, Singh SB, Mallick BN, Muthuraju S, Ilavazhagan G. High altitude memory impairment is due to neuronal apoptosis in hippocampus, cortex and striatum. J Chem Neuroanat 2008;36:227–38.
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Physiology & Behavior journal homepage: www.elsevier.com/locate/phb
Guanfacine ameliorates hypobaric hypoxia induced spatial working memory deficits H. Kauser, S. Sahu, S. Kumar, U. Panjwani ⁎ Defence Institute of Physiology and Allied Sciences (DIPAS), Defence Research and Development Organization (DRDO), Lucknow Road, Timarpur, Delhi 110054, India
H I G H L I G H T S • HH results in PFC dependent cognitive deficits and PFC apoptotic cell death. • GFC administration improves performance of PFC dependent DAT task during the HH. • GFC also protects apoptotic neuronal degeneration in PFC under HH.
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Article history: Received 12 December 2012 Received in revised form 14 May 2013 Accepted 22 October 2013 Keywords: Hypobaric hypoxia Guanfacine Prefrontal cortex Spatial working memory Apoptosis Neurodegeneration
a b s t r a c t Hypobaric hypoxia (HH) observed at high altitude causes mild cognitive impairment specifically affecting attention and working memory. Adrenergic dysregulation and neuronal damage in prefrontal cortex (PFC) has been implicated in hypoxia induced memory deficits. Optimal stimulation of alpha 2A adrenergic receptor in PFC facilitates the spatial working memory (SWM) under the conditions of adrenergic dysregulation. Therefore the present study was designed to test the efficacy of alpha 2A adrenergic agonist, Guanfacine (GFC), to restore HH induced SWM deficits and PFC neuronal damage. The rats were exposed to chronic HH equivalent to 25,000 ft for 7 days in an animal decompression chamber and received daily treatment of GFC at a dose of 1 mg/kg body weight via the intramuscular route during the period of exposure. The cognitive performance was assessed by Delayed Alternation Task (DAT) using T-Maze and PFC neuronal damage was studied by apoptotic and neurodegenerative markers. Percentage of correct choice decreased significantly while perseverative errors showed a significant increase after 7 days HH exposure, GFC significantly ameliorated the SWM deficits and perseveration. There was a marked and significant increase in chromatin condensation, DNA fragmentation, neuronal pyknosis and fluoro Jade positive cells in layer II of the medial PFC in hypoxia exposed group, administration of GFC significantly reduced the magnitude of these changes. Modulation of adrenergic mechanisms by GFC may serve as an effective countermeasure in amelioration of prefrontal deficits and neurodegenerative changes during HH. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Hypobaric hypoxia (HH) encountered during ascent to high altitude is a stressful condition as it is associated with a decreased partial pressure of oxygen leading to reduced oxygen delivery to tissues [1]. Brain in particular is highly vulnerable to such hypoxic stress due to its high oxygen requirement and results in higher order cognitive dysfunctions [2]. The cognitive impairment induced by high altitude exposure draws special concern because these problems compromise a Abbreviations: HH, hypobaric hypoxia; PFC, prefrontal cortex; GFC, Guanfacine; SWM, spatial working memory; DAT, delayed alternation task. ⁎ Corresponding author at: Neurophysiology Division, Defence Institute of Physiology and Allied Sciences (DIPAS), Defence Research and Development Organization (DRDO), Lucknow Road, Timarpur, Delhi-110054, India. Tel.: +91 1123883203; fax: +91 1123914790. E-mail addresses: [email protected], [email protected] (U. Panjwani). 0031-9384/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.physbeh.2013.10.028
person's performance while carrying out highly demanding mental functions requiring attention, vigilance, judgment and working memory. Working memory is thought to lie at the core of many higher order cognitive functions and is defined as the ability to transiently hold information in mind for subsequent manipulation in order to guide goaldirected behavior [3]. The working memory is regulated by prefrontal cortex (PFC) and is created by networks of PFC neurons engaged in recurrent excitation generating persistent activity [4]. The PFC recurrent excitatory firing is profoundly altered by the norepinephrine (NE) and thereby influencing the working memory functions [5]. Chronic exposure to HH is known to cause NE dysregulation [6], working memory deficits [7] and neurodegeneration in brain regions related to learning and memory [8]. This suggests that NE dysregulation under HH may be a possible underlying mechanism leading to cognitive deficits and associated morphological damage. Therefore, targeting NE mechanism in PFC regulating working memory is likely to solve the problems of PFC dysfunctions associated with high altitude ascent.
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Alpha-2 adrenergic agonist is very well known to improve PFC cognitive functions including attention, working memory and behavioral inhibition [9]. Several studies have shown the attenuating influence of alpha-2 adrenergic agonists on the behavioral and morphological consequences in animal model of memory loss [10]. Alpha-2 adrenergic agonist also protects PFC dysfunction and neuronal damage in cerebral ischemia [11]. The neuroprotective effect of alpha-2 adrenergic agonist is exerted by modulation of both presynaptic and postsynaptic mechanisms [12]. Presynaptically, it reduces excitatory neurotransmitter release which is thought to be the underlying cause of hypoxia induced excitotoxicity [13]. Postsynaptic variation includes inhibition of adenylate and guanylate cyclases and thus reduces the neurodegenerative changes induced by excessive intracellular signaling [14]. Moreover, both in vivo and in vitro studies have shown that the beneficial effects of alpha-2 adrenergic agonists are mediated exclusively via the α2A subtype [15]. Although HH induced dysfunction presents a similar picture as seen in other conditions of PFC dysfunction like Schizophrenia [16] and ADHD [17] where Guanfacine, a specific alpha-2A adrenergic agonist proved to be beneficial, its possible beneficial effect in HH has not been examined. Thus, the present study examined the efficacy of Guanfacine (GFC), as a potential therapeutic agent for HH induced PFC cognitive deficit and neuronal damage. 2. Materials and methods
administration. Rats received intra muscular injection of the drug (1 mg/kg body weight), or sterile saline (equal volume) daily during hypoxic exposure for 7 days 15 min before behavioral testing. The 1 mg/kg body weight of the GFC has been reported to be effective to enhance spatial working memory without any sedative, hypotensive or cumulative effects [18,19]. 2.4. Cognitive assessment Rats were trained to learn and perform delayed alternation task (DAT) to test their spatial working memory according to the protocol described by Deacon et al. with some modifications [20]. All animals were maintained on a restricted diet at 80% of their free-feeding weight throughout the experiment. They were initially habituated to a T-maze until they started eating chocolate chips placed in the food wells at the end of each arm. After habituation, training was given until rat's performance stabilized at criterion performance (80% correct choice) on two consecutive sessions (24 h apart). This allowed detection of change in performance with the treatment. At the first trial, rats were given chocolate chips for traversing the runway and entering either arm. Thereafter, for a total of 10 trials per session, they were rewarded only on choosing the maze arm which was not previously visited. Performance was evaluated as the number of trials correct out of 10. Rats were tested 4 times during the HH exposure on testing days i.e. day 1, day 3, day 5 and day 7.
2.1. Animal and experimental protocol 2.5. Tissue processing and microscopy Adult male Sprague–Dawley rats with an average body weight of 240–260 g were used in the present study. Animals were housed in cages (46 cm × 24 cm × 20 cm) with two animals per cage in an air conditioned temperature (22 ± 2 °C) and humidity (55–60%) controlled room with a 12 h light and dark cycles in the animal house of the institute. Rats were provided with food pellet (Lipton India Ltd., India) and water ad libitum until the behavioral study. Experiments to be conducted were approved by the Institutional Animal Ethical Committee. The guidelines documented in the National Institutes of Health's Guide for the Care and Use of Laboratory Animals were followed and all efforts were made to minimize animal suffering. Experimental animals were divided into four groups, Normoxia + Saline (N + SAL, n = 6), Normoxia + Guanfacine (N + GFC, n = 6), Hypoxia + Saline (H + SAL, n = 6) and Hypoxia + Guanfacine (H + GFC, n = 6). On 1st, 3rd, 5th and 7th day of hypoxia exposure, cognitive assessment was done. Guanfacine was administered each day 15 min before cognitive assessment. On the 8th day rats were sacrificed to study their brain morphology.
After HH exposure the animals were perfused transcardially first with ice-cold PBS (0.1 M, pH 7.4) followed by fixation using ice-cold 4% paraformaldehyde (dissolved in 0.1 M PBS, pH 7.4). The brains were dissected out and post-fixed in the same fixative for 24 h at room temperature. The paraformaldehyde-fixed brain were cryoprotected by graded sucrose (10%, 20% and 30%, respectively) solution and 30 μm-thick coronal sections were cut using a cryostat (Leica-3050, Germany) and were placed in 24 well tissue culture plates. Six coronal sections per animal were collected from the PFC at the bregma + 3.20 to + 2.20 mm. The sections were stored at 4 °C until staining. Light microscopy and fluorescence microscopy were performed for visualization. The photomicrographs of the region of interests were captured with a digital camera (Nikon Tokyo Japan) attached to the microscope (Olympus) at ×400 magnification. The neurons were counted from the pyramidal layer II of the medial PFC using ‘Image J’ software (NIMH, U.S.A). The number of neurons was expressed per 55052 mm2 area.
2.2. Exposure to hypobaric hypoxia
2.6. Apoptosis
Exposure to simulated high altitude of 25,000 ft was performed in a specially designed animal decompression chamber (Seven Star, India) operated under a 12 h light–dark cycle which reduced the barometric pressure (atmospheric pressure equivalents to 282 mmHg, PO2 59 mmHg); temperature and relative humidity were maintained at 28–30 °C and 55–60% respectively. The chamber was continuously flushed with fresh air (5.5 l/min) to replenish O2 consumed by rats and remove CO2 produced. The desired altitude (i.e. 25, 000 ft) was attained at an ascent rate of 300 m/min over a period of 20 min. The chamber was brought down to sea level pressure in the morning at 10 o'clock for 1 h every day to replenish food and water, drug treatment and cognitive assessment. Rats were exposed to HH conditions for 7 consecutive days.
2.6.1. Chromatin condensation Hoechst 33342 (Sigma, U.S.A), chromatin dye, freely enters living cells and therefore stains the nuclei of viable cells as well as those undergoing apoptotic or necrotic death. Apoptotic cells were distinguished from viable and necrotic cells by nuclear condensation and fragmentation. Cryostat sections were permeabilized with 0.1% triton in PBS and stained with Hoechst 33342 (10 μg/ml). The stained sections were visualized in UV light and nuclear damage was documented by counting the number of Hoechst positive cells emitting bright fluorescence and exhibiting nuclear morphology.
2.3. Pharmacological intervention Alpha-2A adrenergic agonist, Guanfacine Hydrochloride (Sigma, U.S.A) was dissolved in sterile saline freshly each day before
2.6.2. DNA fragmentation In situ labeling of DNA fragmentation by TUNEL staining was used to study the apoptotic activity. ApopTag Red In Situ Apoptosis Detection Kit (Millipore, U.S.A) was used as per manual instruction for detection of nucleases activated DNA fragments of approximately 200 base pairs. DNA strand breaks were detected by enzymatically labeling the free 3′-OH termini with digoxigenin nucleotides followed by binding
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of fluorescein conjugated anti-digoxigenin antibody. Fluorescent signal was visualized with appropriate excitation/emission filter pairs. The red fluorescence TUNEL positive neurons were counted. 2.7. Neurodegeneration 2.7.1. Neuronal morphology Nissle staining was performed to study the neuronal morphology. The section was immersed in 0.1% cresyl violet (Sigma, U.S.A) for 2 min. Subsequently, it was thoroughly washed and again dehydrated by graded alcohol, cleared, and finally mounted in D.P.X. The small sized, dense, irregularly shaped pyknotic cells were considered as dead and were counted manually in both hemispheres of the medial PFC. 2.7.2. Neuronal degeneration For neurodegeneration study the brain sections were stained with Fluoro Jade-B (Millipore, U.S.A), a poly anionic fluorescence derivative which sensitively and specifically binds to degenerating neurons. The sections were immersed in 1% NaOH in 80% ethanol for 2 min followed by another immersion in 70% ethanol for 2 min and then washed in distilled water for 2 min. The sections were then transferred to fresh 0.06% potassium permanganate solution for 15 min and gently shaken on a rotating platform. This was followed by rinsing in distilled water and transferring to Fluoro Jade B stain where they were gently agitated for 20 min. After staining, the sections were rinsed in distilled water for 2 min, thrice each. Excess water was drained off the slides, and the slides were rapidly dried on a hot plate. The slides were cleared in xylene and then mounted with D.P.X. The sections were visualized using a filter system FITC, and a bright green fluorescence signal indicated degenerating neurons.
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2.8. Statistical analysis All the results were expressed as Mean ± S.E.M. Group differences of behavioral and morphometric data were compared by using oneway ANOVA. Wherever significant differences were found, Bonferroni multiple comparison test was performed as a post hoc analysis to compare the differences between the groups. Difference below or equal the probability level 0.05 was considered statistically significant. 3. Results 3.1. Cognitive assessment 3.1.1. Training Percentage of correct choice was taken as a measure of performance in T maze delayed alternation task (DAT) to test the prefrontal cortical functions. All rats had been trained on this task to an equivalent level of performance. Correct percentage in each group was statistically compared with the baseline value separately in all the groups to illustrate performance over time. With training, correct percentage increased and it is statically significant at last two consecutive sessions (D6, p b 0.001; D7, p b 0.001) of training (Fig. 1A) indicating the learning of DAT during the training period. 3.1.2. Spatial working memory At the end of training, rats were exposed to simulated hypobaric hypoxia equivalent to 25,000 ft for 7 days and were assessed for cognitive ability 4 times over the 7 days. Percentage of correct choice significantly decreased (p b 0.001) after 7 days hypobaric hypoxia exposure when compared to baseline (D0). There is a regular trend in performance impairment in the exposed group during the hypoxia and the change
Fig. 1. Effect of Guanfacine on delayed alteration t-maze task under hypobaric hypoxia. (A) Training of delayed alternation T-Maze task. Percentage of correct choices, an indicator of working memory performance was 80% in the last two consecutive sessions showing learning of the task. (B) Performance of rats during exposure to hypobaric hypoxia for different durations. Percentage of correct choice increased in rats treated with Guanfacine as compared to the hypoxic group in the last two sessions. (C) Working memory performance after 7 days of exposure to hypobaric hypoxia. Performance increased by Guanfacine treatment in rats exposed to 7 days of hypoxia. (D) Perseverance was measured by consecutive errors after 7 days of exposure to hypobaric hypoxia. Consecutive errors increased after hypoxia exposure whereas the errors decreased with Guanfacine treatment during hypoxia. The values represented as Mean ± SEM. *p b 0.05 and ***p b 0.001 versus normoxia and #p b 0.05 and ###p b 0.001 versus hypoxia. Normoxia + Saline (N + SAL), Normoxia + Guanfacine (N + GFC), Hypoxia + Saline (H + SAL) and Hypoxia + Guanfacine (H + GFC).
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was significant at D3 (p b 0.05), D5 (p b 0.01) and D7 (p b 0.001) when compared to D0 (Fig. 1B) indicating consistence deficits on PFC dependent task during the hypoxia exposure. In drug treated group, GFC was administered daily during the hypoxia exposure and in this group performance reached to baseline value (D0) after 7 days of hypobaric hypoxia (Fig. 1C). This amelioration of the PFC deficit by drug administration during the hypoxia exposure is evident at all testing time points i.e. D1, D3, D5 and D7 but it is statistically significant during the two last session (D5, p b 0.5; D7, p b 0.001) of testing (Fig. 1B). Perseveration, defined as the mean number of consecutive errors per test session is another parameter to test PFC functions (Fig. 1D). There was an increase in perseveration in hypoxia exposed rats (p b 0.001) and it was rescued in the hypoxic group treated with the GFC (p b 0.05). These results indicate improvement in the performance of a PFC dependent task by GFC administration during the hypoxia exposure.
3.2. Apoptosis 3.2.1. Chromatin condensation Chronic exposure for 7 days was found to increase chromatin condensation by 11 fold (29.33 ± 1.71 Hoescht Positive Neurons/ 55052 mm2) in medial PFC compared to normoxia group (2.50 ± 0.50, p b 0.001) (Fig. 2A–B). GFC administration decreased chromatin condensation by 3 fold (7.83 ± 0.31) as compared to hypoxia exposed animals (p b 0.001). This indicates the GFC rescue chromatin condensation which is characteristic of cells undergoing apoptosis under HH.
3.2.2. DNA fragmentation A 13 fold (26.33 ± 0.67 TUNEL Positive Neurons/55052 mm2) increase in DNA fragmentation was observed in medial PFC on 7 days of exposure to HH as compared to the normoxic animals (2 ± 0.37, p b 0.001) (Fig. 2C–D). The number of TUNEL positive neurons however decreased by 4 fold (26.33 ± 0.67) on the administration of GFC during HH than hypoxia exposed group (p b 0.001). This result is in validation of the role of GFC under HH to protect neuronal apoptosis in term of DNA fragmentation in the medial PFC. 3.3. Neurodegeneration 3.3.1. Neuronal morphology In case of normoxia group there was no alteration of morphology but exposure to HH showed pyknotic and tangle-like appearance along with large dot like neurons in medial PFC. The pyknotic cell count in medial PFC was increased by 29 fold (26 ± 2.44) after 7 days of hypoxia exposure compared to normoxia group (0.88 ± 0.15, P b 0.001) (Fig. 3A–B). Daily treatment with GFC for hypoxia exposed animals minimized the neuronal damage by 6 fold (4.24 ± 0.44) as compared to animals exposed to HH without treatment (p b 0.001) indicating the neuroprotection of hypoxia induced neurodegeneration in medial PFC by GFC administration. 3.3.2. Neuronal degeneration Exposure to HH for 7 days showed 19 fold increase in Fluoro Jade-B positive neurons (27.33 ± 1.05) in medial PFC as compared to normoxia
Fig. 2. The effect of Guanfacine on hypobaric hypoxia induced apoptosis. (A) Chromatin condensation by Hoechst 33342 staining. (B) Graph showing the change in number of Hoechst positive neurons. (C) DNA fragmentation by TUNEL assay. (D) Graph showing the change in number of TUNEL positive neurons. Normoxia + Saline (N + SAL), Normoxia + Guanfacine (N + GFC), Hypoxia + Saline (H + SAL) and Hypoxia + Guanfacine (H + GFC). Bars represent Mean ± SEM of average of six fields in six individuals of each group. *** denotes p b 0.001 when compared to normoxic group and ### denotes p b 0.001when compared to hypoxic group. The arrows indicate apoptotic neuron (magnification ×400).
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Fig. 3. The effect of guanfacine on hypobaric hypoxia induced Neurodegeneration. (A) Neuronal pyknosis by Nissle staining. (B) Graph showing the change in the number of dead neurons. (C) Neuronal degeneration as is shown by Fluoro Jade B staining. (D) Graph showing the change in number of Fluoro Jade B positive neurons. Normoxia + Saline (N + SAL), Normoxia + Guanfacine (N + GFC), Hypoxia + Saline (H + SAL) and Hypoxia + Guanfacine (H + GFC). Bars represent Mean ± SEM of average of six fields in six individuals of each group. *** denotes p b 0.001 when compared to normoxic group and ### denotes p b 0.001when compared to hypoxic group. The arrows indicate neurodegenerative neuron (magnification ×400).
group (1.42 ± 0.30, p b 0.001) (Fig. 3C–D). The neuronal degeneration in GFC treated hypoxic groups was lowered by 6 fold (4.04 ± 0.20) than animals subjected to similar duration of hypoxia exposure but without drug administration (p b 0.001). This result further validated the neuroprotection of medial PFC neurons by GFC administration during the HH. 4. Discussion The results presented herein demonstrate that exposure to HH impaired spatial working memory performance on the DAT. This is consistent with previous findings showing the working memory impairment under hypoxic condition using different testing paradigms [21]. DAT is a specific test of PFC dependent spatial working memory [22] and working memory has been shown to be highly influenced by NE in PFC [23]. HH induced spatial working memory deficits observed in the present study might also be correlated with dysregulation in NE transmission as in other stressful conditions [24]. Acute stress impairs prefrontal cortical function through a cascade of events: There is a large increase in NE release in the PFC, which in turn leads to high levels of cyclic adenosine monophosphate (cAMP) and protein kinase C (PKC) intracellular signaling, which reduces prefrontal neuronal firing, and rapidly impairs working memory [25]. When stressors are repeated over many days and weeks, there are neurodegenerative changes attributed to enhance intracellular signaling stimulated by altered levels of NE [26]. A systemic administration of GFC (1 mg/kg b.w.), an alpha-2A adrenergic agonist during the hypoxia exposure showed improvement in spatial working
memory and this change was significant after 5 and 7 days of hypoxia exposure when neuronal damage has been reported to be maximum [27]. This indicates that exposure to HH induced a dramatic and progressive impairment of PFC dependent task in vehicle-treated rats, growing more pronounced over the duration of the hypoxia exposure whereas stimulation of alpha-2A receptors with daily GFC treatment rescued DAT performance in exposed rats. The morphological data to study apoptosis and neurodegeneration in medial PFC showed that there was increased cell damage in medial PFC after 7 days of hypoxia exposure. Lesion and pharmacological studies in rodents have implicated the role of medial prefrontal cortex in working memory tasks [28]. Chronic stress severely disrupts two key processes attributed to the PFC: working memory and behavioral flexibility; these behavioral deficits closely correlated with a selective reduction in the volume of the upper prefrontal layers (I and II) [29]. Although hypoxia has been reported to cause spatial working memory impairment, it has not been examined if these deficits could be due to neuronal damage in the PL/IL cortices, structures within the medial prefrontal cortex of rodents that are critical for working memory. The loss of these neurons is expected to have great consequences for cognitive performance. Using a HH animal model, the present study has revealed that the apoptosis and neurodegeneration increased in the PL/IL tissue of exposed animals, which was associated with cognitive deficit. Exposure to HH resulting in a significant reduction in neuronal number may be due to the stress associated with hypoxia [30]. Daily treatment of GFC during the hypoxia exposure rescued the neuronal damage significantly in these brain regions. Interestingly, the behavioral results corroborated
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with the hypoxia induced cortical damage. The beneficial effect of GFC treatment was most pronounced in the late phase of the exposure when morphological changes were observed. These findings reiterate that altered NE transmission contributes to prefrontal dysfunction and neuronal damage, and that stimulation of alpha-2A receptors may provide relief for the working memory deficits observed in HH. 5. Conclusion Modulation of adrenergic mechanisms by GFC may play a role in the amelioration of prefrontal deficits and neurodegenerative changes during hypobaric hypoxia. It appears that the neurodegeneration has a role in cognitive dysfunction and protecting prefrontal regions vulnerable to hypoxia may be a key factor in rescuing prefrontal cortical regulation of behavior and cognition. Therefore we suggest that long lasting PFC dependent cognitive impairment observed during hypobaric hypoxia may be due to neuronal damage in PFC and these devastating changes can be ameliorated by administration of alpha 2A adrenergic agonist. Acknowledgments This study was supported by Defence Research and Development Organization, Ministry of Defence, Government of India and Department of Science and Technology, Government of India. References [1] Savourey G, Launay JC, Besnard Y, Guinet A, Travers S. Normo- and hypobaric hypoxia: are there any physiological differences? Eur J Appl Physiol 2003;89(2):122–6. [2] Lieberman P, Protopapas A, Reed E, Youngs JW, Kanki BG. Cognitive deficits at high altitude. Nature 1994;372:325. [3] Baddeley A. Working memory. Science 1992;255:556–9. [4] Goldman-Rakic PS. Cellular basis of working memory. Neuron 1995;14:477–85. [5] Arnsten AFT. Through the looking glass: differential noradrenergic modulation of prefrontal cortical function. Neural Plast 2000;7:1–2. [6] Saligaut C, Chretien P, Daoust M, Moore N, Boismare F. Dynamic characteristics of dopamine, norepinephrine and serotonin metabolism in axonal endings of the rat hypothalamus and striatum during hypoxia: a study using HPLC with electrochemical detection. Methods Find Exp Clin Pharmacol 1986;8(6):343–9. [7] Kramer AF, Coyne JT, Strayer DL. Cognitive function at high altitude. Hum Factors 1993;35(2):329–44. [8] Maiti P, Singh SB, Mallick BN, Muthuraju S, Ilavazhagan G. High altitude memory impairment is due to neuronal apoptosis in hippocampus, cortex and striatum. J Chem Neuroanat 2008;36:227–38.
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