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The effect of hypoxia at different embryonic ages on impairment of memory ability in chicks
Int. J. Devl Neuroscience 26 (2008) 113–118 www.elsevier.com/locate/ijdevneu
The effect of hypoxia at different embryonic ages on impairment of memory ability in chicks Candice L. Rodricks a,b, Marie E. Gibbs b,c, Graham Jenkin d, Suzanne L. Miller a,d,* a Department of Physiology, Monash University, Clayton, 3800 Victoria, Australia Department of Pharmacology, Monash University, Clayton, 3800 Victoria, Australia c Department of Anatomy and Developmental Biology, Monash University, Clayton, 3800 Victoria, Australia d Department of Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, 3800 Victoria, Australia b
Received 6 June 2007; received in revised form 12 August 2007; accepted 16 August 2007
Abstract Hypoxia during the prenatal period is a principal antecedent to cognitive impairment after birth. In this study we have investigated the duration, severity and timing of acute hypoxia during chick embryonic development to elucidate the relative importance of these factors. Our results show that 24 h of hypoxia (exposure to 14% oxygen) at embryonic day 10 (E10) results in significant impairment of intermediate and long-term memory in the post-hatch chick, which is the same as we observed with 4 days of hypoxia. At E14, 24 h of hypoxia, 5 min of anoxia, but not 1 h of hypoxia, resulted only in impaired long-term memory; the same as 4 days of hypoxia from E14. Corticosterone levels, measured post-hatch as an indicator of a stress response, were significantly elevated in response to E10 hypoxia, and E14 hypoxia (both 1 and 24 h) and anoxia. In a separate experiment we exposed embryos to 24 h of hypoxia from E6 to E16, and found that memory deficits resulted from hypoxia at E9 and E10, and E13–E15, while corticosterone concentrations at hatch were significantly raised following E10–E16 hypoxia. These results demonstrate that the developmental age when the insult occurs determines the nature of the cognitive deficit and, if the severity of the insult is sufficient, then the outcome, or deficits in memory ability, are consistent whether the insult is acute or chronic. Importantly, there are two critical stages in development, which in the chick are around E10 and E14, when acute hypoxia results in significant adverse cognitive effects after hatch. These time-points correspond to two different stages in growth and development. # 2007 ISDN. Published by Elsevier Ltd. All rights reserved. Keywords: Chick; Hypoxia; Memory; Bead discrimination learning; Corticosterone
1. Introduction Compromised prenatal development, particularly episodes of hypoxia that reduce oxygen supply to the fetus, are likely to be the cause of many neurological disorders carried into adulthood, ranging from mild learning difficulties (Gadian et al., 2000; Vargha-Khadem et al., 2003), to attention deficit hyperactivity disorder or cerebral palsy (Gaffney et al., 1994). In humans, prenatal hypoxia may be caused by a range of adverse conditions which include umbilical cord occlusion or through disruption of uteroplacental blood flow (Rees and
* Corresponding author at: Department of Physiology, Building 13F, Monash University, Clayton, 3800 Victoria, Australia. Tel.: +61 3 9905 5130; fax: +61 3 9905 2547. E-mail address: [email protected] (S.L. Miller). 0736-5748/$34.00 # 2007 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2007.08.006
Harding, 2004). In chicks, development occurs without maternal and placental influences and provides a model in which we can assess the nature of the damage caused by hypoxia during embryonic and fetal development on subsequent memory ability after hatch. As chicks are precocial, memory ability can be tested soon after hatch. In normal chicks, three stages of memory are seen in a single trial bead discrimination learning task. The three stages are sequential such that if an early one is absent then the others do not occur. The stages are short-term memory (STM) of 10 min duration, intermediate-term memory (ITM) lasting for a further 40 min and long-term memory (LTM) available from 60 min after training and have been defined on the basis of both behavioural and pharmacological data (Gibbs and Ng, 1977, 1979; Gibbs and Summers, 2002). During incubation, exposure of the chick embryo to hypoxia by experimentally reducing the oxygen supply is detrimental to
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development, affecting both organ and whole body growth (Camm et al., 2001; Dzialowski et al., 2002; Miller et al., 2002). Blood gases are adversely affected (Camm et al., 2000, 2005; Dzialowski et al., 2002) and varying the length or degree of hypoxia results in different outcomes for the embryo and the newly hatched chick. Anoxia (0% oxygen) for 5 min at embryonic day 10 (E10), E13, E16 or E19, results in significantly increased noradrenaline and adrenaline levels 5 min after the insult (Mulder et al., 2000), and hypoxia induced by 50% reduction in surface of the egg for gas exchange results in increased noradrenaline and adrenaline levels at hatch (Camm et al., 2004). A wide range of severities of hypoxic insults (from 10 to 15% ambient oxygen concentration) and durations (from 2 h to 6 days) over early, mid and late gestation demonstrate varying effects on embryonic growth (Altimiras and Phu, 2000; Camm et al., 2001; Dzialowski et al., 2002; Miller et al., 2002). For example, exposure to 6 h of acute hypoxia (10% oxygen) at E4 significantly increased mortality by mid-incubation, but did not affect embryo weight (Altimiras and Phu, 2000), whereas chronic hypoxia from E1–6 or E0–10 (14–15%) decreased body weight at mid-incubation, but there was no longer a difference at hatch (Dzialowski et al., 2002; Miller et al., 2002). These results demonstrate the difficulty in comparing the contributions of these insults to compromised development. In our previous studies we have investigated the effects of a chronic reduction in gas exchange to the developing embryo, by covering half of the egg surface, between incubation days 10– 14 or 14–18 (Camm et al., 2001) and we have compared the results with environmental hypoxia (14% oxygen) over the same timecourse (Rodricks et al., 2004). In the earlier studies hypoxia was achieved by covering the egg with an impermeable membrane (Camm et al., 2005). When eggs were covered from E10 to E14, training at hatch resulted in chicks with poor shortterm and subsequent memory; but if the eggs were covered between E14 and E18 the chicks had good short-term memory but were unable to consolidate memory beyond 30 min (Camm et al., 2001). The nature of the cognitive damage after hatch is different for the two periods of hypoxia. These two gestational periods reflect critical periods in the development of the chick brain, particularly with respect to neuronal and astrocytic growth and differentiation (Sedlacek, 1972; Camm et al., 2005). In the chick, unlike the rodent, there is good temporal resolution between neurogenesis, neural migration, synaptogenesis and synaptic maturation. It is known that neural migration is complete in the chick embryo by E9–10; however, synaptogenesis is not complete until approximately 2 days after hatch (Rostas et al., 1991). This temporal resolution allows the isolation of these events in the study of prenatal insults. The aim of the present study was to use a shorter duration of hypoxia over a wide range of gestational ages to more closely examine the nature of the impaired memory ability and factors which may contribute to poor memory. In the current study, we examined the effects of hypoxia from E6 to E16 of the 21 day incubation period. In addition to examining the effects of prenatal hypoxia on memory processing after hatch, we assessed gross body growth and plasma corticosterone levels after hatch.
2. Experimental procedures All experimental procedures were approved by the Monash University Animal Ethics Committee and comply with the 1997 Australian Code of practice for the care and use of animals for scientific purposes. Fertile eggs from two egg-laying strains of domestic hens (Rhode Island Red-New Hampshire cross and/or White Leghorn-Black Australorp eggs) were obtained from Wagner’s Poultry Farm (Coldstream, Victoria) and incubated for 21 days (E0–21) in domestic self-turning incubators (42 eggs, Brinsea Poly Hatch Incubator) with exposure to 12 h light/dark cycles. Unless otherwise stated, the incubator conditions were maintained at 21% oxygen, 39 8C and 60% humidity (normoxia). Eggs were weighed prior to incubation and treatment groups were weight matched with control groups to 0.1 g. The two strains of chicks were distributed evenly between groups. On E8, eggs were candled and nonfertile eggs were removed from incubation. Chicks hatched inside the incubator and were left to dry (less than 12 h). When the chicks were removed from the incubator, leg rings were attached for identification; they were weighed and then housed in brooders kept at 29 8C with fresh food and water provided daily. Memory testing was performed on the second day post-hatch. Immediately after testing chicks were weighed and then killed by rapid decapitation and blood was immediately collected into heparin coated tubes for corticosterone assay.
2.1. Anoxic and hypoxic insults Hypoxic conditions were induced by running a mixture of nitrogen (1.7 L/ min) and oxygen (3.5 L/min) into a sealed incubator to reduce oxygen levels to 14 0.2%. This mixture was delivered through a sealed beaker with water to moisten the air entering the incubator. Oxygen levels were monitored throughout the hypoxic period using an oxygen analyser (Teledyne). Under hypoxic conditions, eggs were incubated in 14% oxygen, 39 8C and 60% humidity on the day specified, and at other times were incubated in normoxic conditions. Control eggs were simultaneously incubated in a separate incubator under normoxic conditions for each experimental day. Experiment 1: On day 14, hypoxia (14% oxygen) was induced for 1 h (n = 14) or 24 h (n = 15) or anoxia (0% oxygen; n = 12) was induced for 5 min by running nitrogen through the incubator. The 5 min anoxic period commenced once the oxygen levels in the incubator was reduced to 0%, which took approximately 5 min. In a further group, 24 h of hypoxia was carried out at E10 (n = 17). Experiment 2: Eleven separate groups of chick embryos, ranging between E6 and E16 were exposed to hypoxia (14% oxygen) for 24 h (number of chicks per group = 9–22). Due to limitations in the capacity of the incubators, this was performed over five experiments with a matched control group each time the experiment was undertaken.
2.2. Bead discrimination task The one-trial discriminated avoidance bead task was carried out on the second day after hatch, and has been described in detail previously (Gibbs and Summers, 2002). Briefly, chicks were kept in pairs. A chrome bead (2 mm diameter) dipped in water was presented to both chicks one hour before training to familiarise them with the introduction of beads into their box. Thirty minutes later and 30 min before the training trial, a blue and then a red glass bead (4 mm diameter and again tasting of water) were presented to the chicks in two successive 10 s trials, 2.5 min apart. For the training trial, chicks were presented (for 10 s) with a red bead that had been dipped in concentrated methyl anthranilate (100%, Sigma–Aldrich Inc.) a bitter tasting chemical. Prior to training, chicks have no preference for red or blue beads and peck equally at both. The same memory retention is found when chicks are trained on blue beads (Gibbs and Barnett, 1976). To test for retention association of the bitter taste with the red bed, chicks were presented first with a clean dry red bead and then a clean dry blue bead on two 10 s trials, 2.5 min apart. The number of pecks on the coloured beads was recorded for analysis. If chicks did not peck the red bead on training or the blue bead at test they were excluded from the data analysis at the completion of the experiment. The number of chicks excluded at the time of training was never more than one per experimental group, and only 4% of chicks were eliminated because of failing to peck the blue bead on test.
C.L. Rodricks et al. / Int. J. Devl Neuroscience 26 (2008) 113–118 These figures are not different between chicks exposed to prenatal hypoxia and those chicks not treated during embryonic development. The different groups of chicks were identified with leg tags and experiments were run blind as to condition and then decoded at the completion of the experiment. Memory retention was assessed using a discrimination ratio (DR), which is the ratio of the number of pecks at the blue bead to the number of pecks at both red and blue beads. A tendency to avoid the red bead (DR approaches 1.0) indicates memory formation and shows that the chick remembered that the red bead was bitter tasting during training. Equal pecking of the red and blue bead (DR approaches 0.5) indicates no memory. Generally the chicks do not completely avoid the red bead on test even when they remember and the number of pecks given to the blue bead can vary from 1 to 20 in the 10 s test. The use of aversive and non-aversive beads controlled for performance deficits. In Experiment 1, separate groups of chicks were exposed to hypoxia at E14 (5 min anoxia, 1 h or 24 h hypoxia) and at E10 (24 h hypoxia) and were tested for memory at 30 (ITM) or 120 (LTM) min. In Experiment 2, (24 h hypoxia from E6–16) chicks were tested only at 120 min after training.
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hypoxia at E10 resulting in a reduced DR when compared with its control group, Fig. 1A (control versus E10; F(1, 20) = 10.78, p = 0.004). Reduced environmental oxygen at E14 (hypoxia for 1 h or 24 h and anoxia for 5 min) did not result in reduced DR at 30 min ( p > 0.05). When long-term memory was tested at 120 min, the DR of chicks exposed to hypoxia (24 h) at E10 remained significantly reduced (control versus E10; F(1, 20) = 8.83, p = 0.008), Fig. 1B. At 120 min there was also a significant reduction in the DR of chicks exposed to 24 h of hypoxia at E14 (F(3, 46) = 4.109, p = 0.012) and anoxia at E14 ( p = 0.021), but not hypoxia for 1 h, Fig. 1B. At hatch, corticosterone levels were significantly elevated in chicks that had been exposed to hypoxia for 24 h at E10 and also those chicks that were exposed to hypoxia for 1 or 24 h at
2.3. Righting response Following 24 h of hypoxia at E10 or E14, and in matched controls, the righting response (Hill, 1993) was tested after the chicks were removed from the incubator and weighed (Day 1) and then again 24 h later (Day 2). Briefly, the righting response was used to assess the chick’s innate ability to orient itself by placing chicks on their backs and timing the latency taken to return to an upright position. Chicks that failed to return to the upright position were given a latency of 4 s.
2.4. Corticosterone radioimmunoassay Corticosterone levels were measured in 6–8 randomly selected plasma samples. Immediately after memory testing, control and experimental chicks were decapitated after removal from the experimental cage and trunk blood was collected into heparin coated tubes and centrifuged at 3000 rpm for 10 min. The plasma was removed and stored at 20 8C until radioimmunoassay to determine corticosterone levels using a modified version of a described method (Bye et al., 2001). The current method added separate aliquots of buffer, tracer and antiserum to the samples and the 1 h incubation period was removed. The interassay coefficient of variation was 6% and the intra-assay coefficient of variation was 4%. The minimal detectable concentration was 0.28 ng/ml.
2.5. Statistical analysis All data were analysed using the Statistical Package for Social Sciences, Information Analysis systems Inc., Chicago, Illinois, USA (SPSS). One way ANOVA was performed on plasma corticosterone levels and hatch weights comparing experimental values to the appropriate control values. Memory retention levels on the different days of treatment in Experiment 2 were compared with the saline control. Motor testing after hypoxia at E10 or E14 was also analysed by a one way ANOVA. Dunnett’s t-test (two-sided) was used for post hoc analysis. A two-way ANOVA was carried out on ITM and LTM testing in Experiment 1. Simple main effect analysis was used for post hoc testing. Data is presented as mean S.E.M. and significance was accepted at p 0.05.
3. Results 3.1. Experiment 1: different durations of hypoxia or anoxia There were no significant effects of hypoxia or anoxia on hatch weights when compared with control hatch weights (control 43.8 0.48 g, anoxia 43.19 0.46 g, 1 h hypoxia 44.44 0.63 g and 24 h hypoxia 43.49 0.74 g). Memory retention, measured as the discrimination ratio (DR), showed a significant difference at 30 min between groups, with 24 h of
Fig. 1. Memory retention measured as mean discrimination ratio at 30 min (A) or 120 min (B) after 24 h hypoxia (14% oxygen) at E10 and E14. Other groups of chicks received 5 min anoxic or 1 h of hypoxic treatment. (C) Mean plasma corticosterone levels (ng/ml) measured after training. Results expressed as mean S.E.M., numbers per group indicated at the base of bars. *p < 0.05.
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Table 1 The latency (s) taken for chicks to ‘right’ themselves after 24 h hypoxia at E10 or E14 Group
Day 1 test; latency (s)
n
Day 2 test; latency (s)
n
Control E10 E14
1.13 0.05 1.98 0.03* 1.14 0.03
10 35 27
1.22 0.07 2.14 0.08* 1.19 0.01
6 33 31
* p < 0.05, significantly different to controls and chicks exposed to hypoxia at E14.
E14, as well as those chicks exposed to anoxia, Fig. 1C (F(1, 12) = 16.13, p = 0.002). After 24 h hypoxia at E10, chicks were significantly slower to right themselves on day 1 post hatch compared to both control chicks and chicks hypoxic at E14 (F(2, 68) = 10.83, p < 0.001). These chicks remained significantly slower when retested on day 2 than chicks exposed to hypoxia at E14 (F(2, 67) = 5.04, p = 0.009; Table 1). 3.2. Experiment 2: 24 h of hypoxia from E6–16 Experiment 1 demonstrated that the severity and duration of a prenatal insult both contribute to the cognitive outcome and stress response of the hatched chick. To determine the impact of an hypoxic insult at other developmental ages we exposed the
Fig. 2. Effect of hypoxia (24 h at 14% oxygen) imposed on embryos from E6 to E16 on memory at hatch. Control embryos incubated under normoxic conditions. (A) Memory measured as mean discrimination ratio 120 min after training. (B) Mean plasma corticosterone levels (ng/ml) after training. Results expressed as mean S.E.M., with sample sizes on base of bars. *p < 0.05 compared to control.
embryos to 24 h of hypoxia from E6 to E16 and measured memory 120 min after training as well as corticosterone levels. Multiple experiments over time were required for sufficient sample numbers but in each experiment egg weights were always matched to 0.1 g with their own control group. Overall, the egg weight varied from 59.17 0.15 g to 67.61 0.22 g, however, when compared to their own control group, there was no effect of exposure to hypoxia on hatch weight at any embryonic age studied. Exposing the developing chick to 24 h of hypoxia during incubation demonstrated a significant effect on memory ability after hatch. Overall we observed a significant main treatment effect (F(11, 171) = 7.10, p < 0.01) and Dunnett’s test revealed a decrease in DR when chick embryos were exposed to hypoxia at day E9 ( p < 0.001), E10 ( p < 0.005), E13 ( p < 0.007), E14 ( p = 0.003) and E15 ( p = 0.003). However, there was no effect on memory when hypoxia was imposed at E6–E8, E11, E12 or E16 (Fig. 2A). Plasma corticosterone levels at hatch were not significantly raised after hypoxia from E6 to E9, but there was a significant elevation in levels following hypoxia at all times from E10 to E16 (F(11, 60) = 21.99, p < 0.01) (Fig. 2B). 4. Discussion An acute period of hypoxia or anoxia during chick embryonic development was detrimental to cognitive function after hatch, with the onset of the hypoxic period an important determinant in functional outcome. The chick is a good animal model in which to examine the effects of prenatal hypoxia and other developmental influences on subsequent cognitive abilities, since the direct effect of reducing oxygen supply to the fetus can be achieved, without confounding maternal or placental influences. Cognitive function can readily be assessed after hatch using discriminated bead avoidance training. Twenty four hours of hypoxia at E10 or at E14 of chick embryonic development impaired memory formation in a manner identical to that of 4 days of hypoxia (Camm et al., 2001; Rodricks et al., 2004). In previous experiments we showed that 4 days of hypoxia starting at E10 resulted in chicks with poor short-term memory, whereas hypoxia starting at E14 resulted in chicks which did not consolidate memory after 30 min. In the current study, memory at 30 and 120 min was adversely affected in response to 24 h of hypoxia at E10. After 1 or 4 days of hypoxia starting at E10 (Camm et al., 2001; C.R. Rodricks and other authors, unpublished) memory was impaired 10 min after training, and as the various stages of memory are sequentially dependent upon each other, the other stages of memory did not develop. When the hypoxic insult is at E14, 24 h but not 1 h of hypoxia compromised long-term memory processing in the chick, but memory was good at 30 min, thus demonstrating that intermediate memory was unaffected. These results are the same as we found with 4 days of hypoxia beginning on E14 (Camm et al., 2001; Rodricks et al., 2004). Five minutes exposure to 100% nitrogen in the incubator also resulted in impaired memory consolidation, with deficits in long-term
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memory only. It is unlikely that the oxygen concentration in the egg will reach zero in 5 min, but this reduction was sufficient to impair memory. We are exploring the duration of hypoxia that is required to produce memory deficits, and the shorter the time that we use, the more exactly we can determine the cellular processes that are affected in the brain. These results show that the developmental age when the insult occurs determines the nature of the cognitive deficit and, if the severity of the insult is sufficient, then the outcome, or deficits in memory ability, are the same regardless of whether the insult is acute or chronic. The reduction in oxygen availability is likely to stress the embryo. This idea is supported by the acute increase in plasma noradrenaline levels that occurs within minutes of the anoxic or hypoxic insult (Mulder et al., 2000), and persist at hatch (Camm et al., 2004). Plasma corticosterone levels are also a measure of embryonic stress. In the chick, corticosterone is produced by the adrenal gland from E8 onwards and sympathetic innervation of the adrenal gland occurs at E14 (Romanoff, 1960), so elevated circulating corticosterone levels after hatch may reflect prenatal stress. Acute or chronically raised corticosterone levels adversely affect cognitive function (Sandi and Rose, 1997; Sandi, 1998), but the mechanisms of action may differ. In the present study, 1 or 24 h of hypoxia at E10 or E14, as well as 5 min of anoxia all resulted in significantly elevated plasma corticosterone levels at hatch compared to control chicks. Interestingly, 1 h of hypoxia at E14 caused raised corticosterone levels at hatch, but did not result in memory deficits. This suggests that raised corticosterone per se cannot be the direct cause of the memory deficits. In the longitudinal study of hypoxia from E6 to E16, corticosterone levels remained elevated in response to all insults from E10 onwards, but memory was impaired only following E9 and E10, and E13– E15 hypoxia, which is also indicative that raised corticosterone at hatch does not directly cause impaired memory. In addition to raised corticosterone at hatch, we have previously demonstrated increased corticosterone levels in ovo. After 24 h of hypoxia at E10, plasma corticosterone levels were increased at E11 and E15, and after hypoxia at E14, the hormone levels were raised at E15 and E17 (Rodricks et al., 2006). The observed lack of relationship between chronically raised corticosterone levels at hatch and memory impairment suggests that there is not a direct causal relationship between circulating corticosterone concentrations at hatch and posthatch memory ability. There may be a resetting of the HPA axis which could alter the responsiveness to a further stressor. For example, in fetal sheep it has been shown that acute glucocorticoid exposure results in an alteration of the basal set-point of the HPA axis and an enhanced HPA axis response to subsequent acute hypoxia (Fletcher et al., 2004). This is relevant for the current studies in developing chicks, since the embryos experience a period of hypoxia towards the end of incubation prior to hatch, and then are likely to experience further stress after hatch when handling and testing take place. Within the experiments presented here, the comparison of corticosterone levels between chicks is relative as handling and decapitation will raise corticosterone levels to some degree, but
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all chicks were handled in the same way. We hypothesise that it is the acute increase in circulating corticosterone or noradrenaline at the onset of the hypoxic or anoxic insult that leads to memory impairment after hatch. In support of this, we have recently shown that a single injection of corticosterone (0.2 nmol/egg) onto the chorioallantoic membrane at E10 or E14 is sufficient to mimic the adverse effects of hypoxia on memory ability (Rodricks et al., 2006), as does an injection of noradrenaline at E14 (M.E. Gibbs unpublished). The mechanisms of action of acute prenatal hypoxia, corticosterone or noradrenaline which go on to result in poor memory ability after hatch are not currently known and require further investigation. With respect to the cognitive outcome, the important finding of this study is that there are two vulnerable periods around E10 and E14 during development when the hypoxic insult impacts on subsequent memory processing. Hypoxia at E11 and E12 did not have any subsequent effect of memory, which suggests that hypoxia at E10 and E14 are affecting discrete events in brain development. Indeed, two major developmental milestones occur at E10 and E14. At E10 neuronal migration is essentially complete, but axonal outgrowth, dendritic expansion and synapse formation is just beginning (Tsai et al., 1981). The appearance of astroctytes (as opposed to radial glia), does not occur until E13 or E14 (Striedter and Beydler, 1997; Camm et al., 2005). This developmental distinction suggests that although the insult around E10 may be linked to neuronal development, the insult around E14 could affect astrocytic or neuronal development, or a combination of the two. One may argue that hypoxia causes a deficiency in the ability of the chicks to visually discriminate colours, however, the finding that memory deficits induced by hypoxia can be rescued (Camm et al., 2004), demonstrates that the chick’s ability to discriminate between red and blue is not affected, and thus sight is not affected. Hypoxia on E10 resulted in mild impairment of the righting response at 1 and 2 days after hatch, an effect not apparent after hypoxia at E14. In the past, the righting response has been used as a quantitative indicator of the motor ability of newly hatched chicks when embryos were malnourished prior to incubation (Hill, 1993), which may suggest that E10 hypoxia alters neuromotor function. In fact, the unchanged pecking of the test beads in the discriminated bead task suggests that there is not a motor impairment. The test is however quite similar to a tonic immobility test which assesses the defensive behaviour of animals (including chicks) and therefore may be measuring other aspects of behavioural response, such as fear (Jones, 1989). This result requires further investigation. In conclusion, the present study demonstrates that 24 h of hypoxia at E10 in the developing chick results in the same memory impairments as 4 days of hypoxia beginning on E10. At E14, hypoxia for 24 h or anoxia for 5 min, but not hypoxia for 1 h, produces the same cognitive deficits as 4 days of hypoxia. Hypoxia at E10 adversely affects intermediate and thus long-term memory, whereas insults at E14 only affect consolidation into long-term memory. This shows that if the hypoxic insult is of sufficient severity, then the developmental age at the time of the insult determines the nature of the
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memory deficit and the functional outcome is consistent whether the insult is acute or chronic. Our results also demonstrate that there are two important and distinct developmental stages when hypoxia produces cognitive deficits after hatch, around day E10 and E14, which are likely to correspond with different structural processes occurring at these times. References Altimiras, J., Phu, L., 2000. Lack of physiological plasticity in the early chicken embryo exposed to acute hypoxia. J. Exp. Zool. 286, 450–456. Bye, N., Zeiba, M., Wreford, N.G., Nichols, N.R., 2001. Resistance of the dentate gyrus and induced apoptosis during aging is associated with increases in transforming growth factor B1 messenger RNA. Neuroscience 105, 853–862. Camm, E.J., Gibbs, M.E., Cock, M.L., Rees, S.M., Harding, R., 2000. Assessment of learning ability and behaviour in low birthweight lambs following intrauterine growth restriction. Reprod. Fertil. Dev. 12, 165–172. Camm, E.J., Gibbs, M.E., Harding, R., 2001. Restriction of prenatal gas exchange impairs memory consolidation in the chick. Brain Res. Dev. Brain Res. 132, 141–150. Camm, E.J., Gibbs, M.E., Harding, R., Mulder, T., Rees, S.M., 2005. Prental hypoxia impairs memory function but does not result in overt structural alterations in the postnatal chick brain. Brain Res. Dev. Brain Res. 160, 9–18. Camm, E.J., Harding, R., Lambert, G., Gibbs, M.E., 2004. The involvement of catecholamines in memory impairment induced by prenatal hypoxia. Neuroscience 128, 545–553. Dzialowski, E.M., von Plettenberg, D., Elmonoufy, N.A., BW, W., 2002. Chronic hypoxia alters the physiological and morphological trajectories of developing chick embryos. Comp. Biochem. Physiol.: A Mol. Integr. Physiol. 131, 713–724. Fletcher, A.J., Ma, X.H., Wu, W.X., Nathanielsz, P.W., McGarrigle, H.H., Fowden, A.L., Giussani, D.A., 2004. Antenatal glucocorticoids reset the level of baseline and hypoxemia-induced pituitary-adrenal activity in the sheep fetus during late gestation. Am. J. Physiol. Endocrinol. Metab. 286, E311–E319. Gadian, D.G., Aicardi, J., Watkins, K.E., Porter, D.A., Mishkin, M., VarghaKhadem, F., 2000. Developmental amnesia associated with early hypoxicischaemic injury. Brain 123, 499–507. Gaffney, G., Sellers, V., Squier, M., Johnson, A., 1994. Case-control study of intrapartum care, cerebral palsy, and perinatal death. BMJ 743–750. Gibbs, M.E., Barnett, J.M., 1976. Drug effects on successive discrimination learning in young chickens. Brain Res. Bull. 1, 295–299.
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The effect of hypoxia at different embryonic ages on impairment of memory ability in chicks Candice L. Rodricks a,b, Marie E. Gibbs b,c, Graham Jenkin d, Suzanne L. Miller a,d,* a Department of Physiology, Monash University, Clayton, 3800 Victoria, Australia Department of Pharmacology, Monash University, Clayton, 3800 Victoria, Australia c Department of Anatomy and Developmental Biology, Monash University, Clayton, 3800 Victoria, Australia d Department of Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, 3800 Victoria, Australia b
Received 6 June 2007; received in revised form 12 August 2007; accepted 16 August 2007
Abstract Hypoxia during the prenatal period is a principal antecedent to cognitive impairment after birth. In this study we have investigated the duration, severity and timing of acute hypoxia during chick embryonic development to elucidate the relative importance of these factors. Our results show that 24 h of hypoxia (exposure to 14% oxygen) at embryonic day 10 (E10) results in significant impairment of intermediate and long-term memory in the post-hatch chick, which is the same as we observed with 4 days of hypoxia. At E14, 24 h of hypoxia, 5 min of anoxia, but not 1 h of hypoxia, resulted only in impaired long-term memory; the same as 4 days of hypoxia from E14. Corticosterone levels, measured post-hatch as an indicator of a stress response, were significantly elevated in response to E10 hypoxia, and E14 hypoxia (both 1 and 24 h) and anoxia. In a separate experiment we exposed embryos to 24 h of hypoxia from E6 to E16, and found that memory deficits resulted from hypoxia at E9 and E10, and E13–E15, while corticosterone concentrations at hatch were significantly raised following E10–E16 hypoxia. These results demonstrate that the developmental age when the insult occurs determines the nature of the cognitive deficit and, if the severity of the insult is sufficient, then the outcome, or deficits in memory ability, are consistent whether the insult is acute or chronic. Importantly, there are two critical stages in development, which in the chick are around E10 and E14, when acute hypoxia results in significant adverse cognitive effects after hatch. These time-points correspond to two different stages in growth and development. # 2007 ISDN. Published by Elsevier Ltd. All rights reserved. Keywords: Chick; Hypoxia; Memory; Bead discrimination learning; Corticosterone
1. Introduction Compromised prenatal development, particularly episodes of hypoxia that reduce oxygen supply to the fetus, are likely to be the cause of many neurological disorders carried into adulthood, ranging from mild learning difficulties (Gadian et al., 2000; Vargha-Khadem et al., 2003), to attention deficit hyperactivity disorder or cerebral palsy (Gaffney et al., 1994). In humans, prenatal hypoxia may be caused by a range of adverse conditions which include umbilical cord occlusion or through disruption of uteroplacental blood flow (Rees and
* Corresponding author at: Department of Physiology, Building 13F, Monash University, Clayton, 3800 Victoria, Australia. Tel.: +61 3 9905 5130; fax: +61 3 9905 2547. E-mail address: [email protected] (S.L. Miller). 0736-5748/$34.00 # 2007 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2007.08.006
Harding, 2004). In chicks, development occurs without maternal and placental influences and provides a model in which we can assess the nature of the damage caused by hypoxia during embryonic and fetal development on subsequent memory ability after hatch. As chicks are precocial, memory ability can be tested soon after hatch. In normal chicks, three stages of memory are seen in a single trial bead discrimination learning task. The three stages are sequential such that if an early one is absent then the others do not occur. The stages are short-term memory (STM) of 10 min duration, intermediate-term memory (ITM) lasting for a further 40 min and long-term memory (LTM) available from 60 min after training and have been defined on the basis of both behavioural and pharmacological data (Gibbs and Ng, 1977, 1979; Gibbs and Summers, 2002). During incubation, exposure of the chick embryo to hypoxia by experimentally reducing the oxygen supply is detrimental to
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development, affecting both organ and whole body growth (Camm et al., 2001; Dzialowski et al., 2002; Miller et al., 2002). Blood gases are adversely affected (Camm et al., 2000, 2005; Dzialowski et al., 2002) and varying the length or degree of hypoxia results in different outcomes for the embryo and the newly hatched chick. Anoxia (0% oxygen) for 5 min at embryonic day 10 (E10), E13, E16 or E19, results in significantly increased noradrenaline and adrenaline levels 5 min after the insult (Mulder et al., 2000), and hypoxia induced by 50% reduction in surface of the egg for gas exchange results in increased noradrenaline and adrenaline levels at hatch (Camm et al., 2004). A wide range of severities of hypoxic insults (from 10 to 15% ambient oxygen concentration) and durations (from 2 h to 6 days) over early, mid and late gestation demonstrate varying effects on embryonic growth (Altimiras and Phu, 2000; Camm et al., 2001; Dzialowski et al., 2002; Miller et al., 2002). For example, exposure to 6 h of acute hypoxia (10% oxygen) at E4 significantly increased mortality by mid-incubation, but did not affect embryo weight (Altimiras and Phu, 2000), whereas chronic hypoxia from E1–6 or E0–10 (14–15%) decreased body weight at mid-incubation, but there was no longer a difference at hatch (Dzialowski et al., 2002; Miller et al., 2002). These results demonstrate the difficulty in comparing the contributions of these insults to compromised development. In our previous studies we have investigated the effects of a chronic reduction in gas exchange to the developing embryo, by covering half of the egg surface, between incubation days 10– 14 or 14–18 (Camm et al., 2001) and we have compared the results with environmental hypoxia (14% oxygen) over the same timecourse (Rodricks et al., 2004). In the earlier studies hypoxia was achieved by covering the egg with an impermeable membrane (Camm et al., 2005). When eggs were covered from E10 to E14, training at hatch resulted in chicks with poor shortterm and subsequent memory; but if the eggs were covered between E14 and E18 the chicks had good short-term memory but were unable to consolidate memory beyond 30 min (Camm et al., 2001). The nature of the cognitive damage after hatch is different for the two periods of hypoxia. These two gestational periods reflect critical periods in the development of the chick brain, particularly with respect to neuronal and astrocytic growth and differentiation (Sedlacek, 1972; Camm et al., 2005). In the chick, unlike the rodent, there is good temporal resolution between neurogenesis, neural migration, synaptogenesis and synaptic maturation. It is known that neural migration is complete in the chick embryo by E9–10; however, synaptogenesis is not complete until approximately 2 days after hatch (Rostas et al., 1991). This temporal resolution allows the isolation of these events in the study of prenatal insults. The aim of the present study was to use a shorter duration of hypoxia over a wide range of gestational ages to more closely examine the nature of the impaired memory ability and factors which may contribute to poor memory. In the current study, we examined the effects of hypoxia from E6 to E16 of the 21 day incubation period. In addition to examining the effects of prenatal hypoxia on memory processing after hatch, we assessed gross body growth and plasma corticosterone levels after hatch.
2. Experimental procedures All experimental procedures were approved by the Monash University Animal Ethics Committee and comply with the 1997 Australian Code of practice for the care and use of animals for scientific purposes. Fertile eggs from two egg-laying strains of domestic hens (Rhode Island Red-New Hampshire cross and/or White Leghorn-Black Australorp eggs) were obtained from Wagner’s Poultry Farm (Coldstream, Victoria) and incubated for 21 days (E0–21) in domestic self-turning incubators (42 eggs, Brinsea Poly Hatch Incubator) with exposure to 12 h light/dark cycles. Unless otherwise stated, the incubator conditions were maintained at 21% oxygen, 39 8C and 60% humidity (normoxia). Eggs were weighed prior to incubation and treatment groups were weight matched with control groups to 0.1 g. The two strains of chicks were distributed evenly between groups. On E8, eggs were candled and nonfertile eggs were removed from incubation. Chicks hatched inside the incubator and were left to dry (less than 12 h). When the chicks were removed from the incubator, leg rings were attached for identification; they were weighed and then housed in brooders kept at 29 8C with fresh food and water provided daily. Memory testing was performed on the second day post-hatch. Immediately after testing chicks were weighed and then killed by rapid decapitation and blood was immediately collected into heparin coated tubes for corticosterone assay.
2.1. Anoxic and hypoxic insults Hypoxic conditions were induced by running a mixture of nitrogen (1.7 L/ min) and oxygen (3.5 L/min) into a sealed incubator to reduce oxygen levels to 14 0.2%. This mixture was delivered through a sealed beaker with water to moisten the air entering the incubator. Oxygen levels were monitored throughout the hypoxic period using an oxygen analyser (Teledyne). Under hypoxic conditions, eggs were incubated in 14% oxygen, 39 8C and 60% humidity on the day specified, and at other times were incubated in normoxic conditions. Control eggs were simultaneously incubated in a separate incubator under normoxic conditions for each experimental day. Experiment 1: On day 14, hypoxia (14% oxygen) was induced for 1 h (n = 14) or 24 h (n = 15) or anoxia (0% oxygen; n = 12) was induced for 5 min by running nitrogen through the incubator. The 5 min anoxic period commenced once the oxygen levels in the incubator was reduced to 0%, which took approximately 5 min. In a further group, 24 h of hypoxia was carried out at E10 (n = 17). Experiment 2: Eleven separate groups of chick embryos, ranging between E6 and E16 were exposed to hypoxia (14% oxygen) for 24 h (number of chicks per group = 9–22). Due to limitations in the capacity of the incubators, this was performed over five experiments with a matched control group each time the experiment was undertaken.
2.2. Bead discrimination task The one-trial discriminated avoidance bead task was carried out on the second day after hatch, and has been described in detail previously (Gibbs and Summers, 2002). Briefly, chicks were kept in pairs. A chrome bead (2 mm diameter) dipped in water was presented to both chicks one hour before training to familiarise them with the introduction of beads into their box. Thirty minutes later and 30 min before the training trial, a blue and then a red glass bead (4 mm diameter and again tasting of water) were presented to the chicks in two successive 10 s trials, 2.5 min apart. For the training trial, chicks were presented (for 10 s) with a red bead that had been dipped in concentrated methyl anthranilate (100%, Sigma–Aldrich Inc.) a bitter tasting chemical. Prior to training, chicks have no preference for red or blue beads and peck equally at both. The same memory retention is found when chicks are trained on blue beads (Gibbs and Barnett, 1976). To test for retention association of the bitter taste with the red bed, chicks were presented first with a clean dry red bead and then a clean dry blue bead on two 10 s trials, 2.5 min apart. The number of pecks on the coloured beads was recorded for analysis. If chicks did not peck the red bead on training or the blue bead at test they were excluded from the data analysis at the completion of the experiment. The number of chicks excluded at the time of training was never more than one per experimental group, and only 4% of chicks were eliminated because of failing to peck the blue bead on test.
C.L. Rodricks et al. / Int. J. Devl Neuroscience 26 (2008) 113–118 These figures are not different between chicks exposed to prenatal hypoxia and those chicks not treated during embryonic development. The different groups of chicks were identified with leg tags and experiments were run blind as to condition and then decoded at the completion of the experiment. Memory retention was assessed using a discrimination ratio (DR), which is the ratio of the number of pecks at the blue bead to the number of pecks at both red and blue beads. A tendency to avoid the red bead (DR approaches 1.0) indicates memory formation and shows that the chick remembered that the red bead was bitter tasting during training. Equal pecking of the red and blue bead (DR approaches 0.5) indicates no memory. Generally the chicks do not completely avoid the red bead on test even when they remember and the number of pecks given to the blue bead can vary from 1 to 20 in the 10 s test. The use of aversive and non-aversive beads controlled for performance deficits. In Experiment 1, separate groups of chicks were exposed to hypoxia at E14 (5 min anoxia, 1 h or 24 h hypoxia) and at E10 (24 h hypoxia) and were tested for memory at 30 (ITM) or 120 (LTM) min. In Experiment 2, (24 h hypoxia from E6–16) chicks were tested only at 120 min after training.
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hypoxia at E10 resulting in a reduced DR when compared with its control group, Fig. 1A (control versus E10; F(1, 20) = 10.78, p = 0.004). Reduced environmental oxygen at E14 (hypoxia for 1 h or 24 h and anoxia for 5 min) did not result in reduced DR at 30 min ( p > 0.05). When long-term memory was tested at 120 min, the DR of chicks exposed to hypoxia (24 h) at E10 remained significantly reduced (control versus E10; F(1, 20) = 8.83, p = 0.008), Fig. 1B. At 120 min there was also a significant reduction in the DR of chicks exposed to 24 h of hypoxia at E14 (F(3, 46) = 4.109, p = 0.012) and anoxia at E14 ( p = 0.021), but not hypoxia for 1 h, Fig. 1B. At hatch, corticosterone levels were significantly elevated in chicks that had been exposed to hypoxia for 24 h at E10 and also those chicks that were exposed to hypoxia for 1 or 24 h at
2.3. Righting response Following 24 h of hypoxia at E10 or E14, and in matched controls, the righting response (Hill, 1993) was tested after the chicks were removed from the incubator and weighed (Day 1) and then again 24 h later (Day 2). Briefly, the righting response was used to assess the chick’s innate ability to orient itself by placing chicks on their backs and timing the latency taken to return to an upright position. Chicks that failed to return to the upright position were given a latency of 4 s.
2.4. Corticosterone radioimmunoassay Corticosterone levels were measured in 6–8 randomly selected plasma samples. Immediately after memory testing, control and experimental chicks were decapitated after removal from the experimental cage and trunk blood was collected into heparin coated tubes and centrifuged at 3000 rpm for 10 min. The plasma was removed and stored at 20 8C until radioimmunoassay to determine corticosterone levels using a modified version of a described method (Bye et al., 2001). The current method added separate aliquots of buffer, tracer and antiserum to the samples and the 1 h incubation period was removed. The interassay coefficient of variation was 6% and the intra-assay coefficient of variation was 4%. The minimal detectable concentration was 0.28 ng/ml.
2.5. Statistical analysis All data were analysed using the Statistical Package for Social Sciences, Information Analysis systems Inc., Chicago, Illinois, USA (SPSS). One way ANOVA was performed on plasma corticosterone levels and hatch weights comparing experimental values to the appropriate control values. Memory retention levels on the different days of treatment in Experiment 2 were compared with the saline control. Motor testing after hypoxia at E10 or E14 was also analysed by a one way ANOVA. Dunnett’s t-test (two-sided) was used for post hoc analysis. A two-way ANOVA was carried out on ITM and LTM testing in Experiment 1. Simple main effect analysis was used for post hoc testing. Data is presented as mean S.E.M. and significance was accepted at p 0.05.
3. Results 3.1. Experiment 1: different durations of hypoxia or anoxia There were no significant effects of hypoxia or anoxia on hatch weights when compared with control hatch weights (control 43.8 0.48 g, anoxia 43.19 0.46 g, 1 h hypoxia 44.44 0.63 g and 24 h hypoxia 43.49 0.74 g). Memory retention, measured as the discrimination ratio (DR), showed a significant difference at 30 min between groups, with 24 h of
Fig. 1. Memory retention measured as mean discrimination ratio at 30 min (A) or 120 min (B) after 24 h hypoxia (14% oxygen) at E10 and E14. Other groups of chicks received 5 min anoxic or 1 h of hypoxic treatment. (C) Mean plasma corticosterone levels (ng/ml) measured after training. Results expressed as mean S.E.M., numbers per group indicated at the base of bars. *p < 0.05.
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Table 1 The latency (s) taken for chicks to ‘right’ themselves after 24 h hypoxia at E10 or E14 Group
Day 1 test; latency (s)
n
Day 2 test; latency (s)
n
Control E10 E14
1.13 0.05 1.98 0.03* 1.14 0.03
10 35 27
1.22 0.07 2.14 0.08* 1.19 0.01
6 33 31
* p < 0.05, significantly different to controls and chicks exposed to hypoxia at E14.
E14, as well as those chicks exposed to anoxia, Fig. 1C (F(1, 12) = 16.13, p = 0.002). After 24 h hypoxia at E10, chicks were significantly slower to right themselves on day 1 post hatch compared to both control chicks and chicks hypoxic at E14 (F(2, 68) = 10.83, p < 0.001). These chicks remained significantly slower when retested on day 2 than chicks exposed to hypoxia at E14 (F(2, 67) = 5.04, p = 0.009; Table 1). 3.2. Experiment 2: 24 h of hypoxia from E6–16 Experiment 1 demonstrated that the severity and duration of a prenatal insult both contribute to the cognitive outcome and stress response of the hatched chick. To determine the impact of an hypoxic insult at other developmental ages we exposed the
Fig. 2. Effect of hypoxia (24 h at 14% oxygen) imposed on embryos from E6 to E16 on memory at hatch. Control embryos incubated under normoxic conditions. (A) Memory measured as mean discrimination ratio 120 min after training. (B) Mean plasma corticosterone levels (ng/ml) after training. Results expressed as mean S.E.M., with sample sizes on base of bars. *p < 0.05 compared to control.
embryos to 24 h of hypoxia from E6 to E16 and measured memory 120 min after training as well as corticosterone levels. Multiple experiments over time were required for sufficient sample numbers but in each experiment egg weights were always matched to 0.1 g with their own control group. Overall, the egg weight varied from 59.17 0.15 g to 67.61 0.22 g, however, when compared to their own control group, there was no effect of exposure to hypoxia on hatch weight at any embryonic age studied. Exposing the developing chick to 24 h of hypoxia during incubation demonstrated a significant effect on memory ability after hatch. Overall we observed a significant main treatment effect (F(11, 171) = 7.10, p < 0.01) and Dunnett’s test revealed a decrease in DR when chick embryos were exposed to hypoxia at day E9 ( p < 0.001), E10 ( p < 0.005), E13 ( p < 0.007), E14 ( p = 0.003) and E15 ( p = 0.003). However, there was no effect on memory when hypoxia was imposed at E6–E8, E11, E12 or E16 (Fig. 2A). Plasma corticosterone levels at hatch were not significantly raised after hypoxia from E6 to E9, but there was a significant elevation in levels following hypoxia at all times from E10 to E16 (F(11, 60) = 21.99, p < 0.01) (Fig. 2B). 4. Discussion An acute period of hypoxia or anoxia during chick embryonic development was detrimental to cognitive function after hatch, with the onset of the hypoxic period an important determinant in functional outcome. The chick is a good animal model in which to examine the effects of prenatal hypoxia and other developmental influences on subsequent cognitive abilities, since the direct effect of reducing oxygen supply to the fetus can be achieved, without confounding maternal or placental influences. Cognitive function can readily be assessed after hatch using discriminated bead avoidance training. Twenty four hours of hypoxia at E10 or at E14 of chick embryonic development impaired memory formation in a manner identical to that of 4 days of hypoxia (Camm et al., 2001; Rodricks et al., 2004). In previous experiments we showed that 4 days of hypoxia starting at E10 resulted in chicks with poor short-term memory, whereas hypoxia starting at E14 resulted in chicks which did not consolidate memory after 30 min. In the current study, memory at 30 and 120 min was adversely affected in response to 24 h of hypoxia at E10. After 1 or 4 days of hypoxia starting at E10 (Camm et al., 2001; C.R. Rodricks and other authors, unpublished) memory was impaired 10 min after training, and as the various stages of memory are sequentially dependent upon each other, the other stages of memory did not develop. When the hypoxic insult is at E14, 24 h but not 1 h of hypoxia compromised long-term memory processing in the chick, but memory was good at 30 min, thus demonstrating that intermediate memory was unaffected. These results are the same as we found with 4 days of hypoxia beginning on E14 (Camm et al., 2001; Rodricks et al., 2004). Five minutes exposure to 100% nitrogen in the incubator also resulted in impaired memory consolidation, with deficits in long-term
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memory only. It is unlikely that the oxygen concentration in the egg will reach zero in 5 min, but this reduction was sufficient to impair memory. We are exploring the duration of hypoxia that is required to produce memory deficits, and the shorter the time that we use, the more exactly we can determine the cellular processes that are affected in the brain. These results show that the developmental age when the insult occurs determines the nature of the cognitive deficit and, if the severity of the insult is sufficient, then the outcome, or deficits in memory ability, are the same regardless of whether the insult is acute or chronic. The reduction in oxygen availability is likely to stress the embryo. This idea is supported by the acute increase in plasma noradrenaline levels that occurs within minutes of the anoxic or hypoxic insult (Mulder et al., 2000), and persist at hatch (Camm et al., 2004). Plasma corticosterone levels are also a measure of embryonic stress. In the chick, corticosterone is produced by the adrenal gland from E8 onwards and sympathetic innervation of the adrenal gland occurs at E14 (Romanoff, 1960), so elevated circulating corticosterone levels after hatch may reflect prenatal stress. Acute or chronically raised corticosterone levels adversely affect cognitive function (Sandi and Rose, 1997; Sandi, 1998), but the mechanisms of action may differ. In the present study, 1 or 24 h of hypoxia at E10 or E14, as well as 5 min of anoxia all resulted in significantly elevated plasma corticosterone levels at hatch compared to control chicks. Interestingly, 1 h of hypoxia at E14 caused raised corticosterone levels at hatch, but did not result in memory deficits. This suggests that raised corticosterone per se cannot be the direct cause of the memory deficits. In the longitudinal study of hypoxia from E6 to E16, corticosterone levels remained elevated in response to all insults from E10 onwards, but memory was impaired only following E9 and E10, and E13– E15 hypoxia, which is also indicative that raised corticosterone at hatch does not directly cause impaired memory. In addition to raised corticosterone at hatch, we have previously demonstrated increased corticosterone levels in ovo. After 24 h of hypoxia at E10, plasma corticosterone levels were increased at E11 and E15, and after hypoxia at E14, the hormone levels were raised at E15 and E17 (Rodricks et al., 2006). The observed lack of relationship between chronically raised corticosterone levels at hatch and memory impairment suggests that there is not a direct causal relationship between circulating corticosterone concentrations at hatch and posthatch memory ability. There may be a resetting of the HPA axis which could alter the responsiveness to a further stressor. For example, in fetal sheep it has been shown that acute glucocorticoid exposure results in an alteration of the basal set-point of the HPA axis and an enhanced HPA axis response to subsequent acute hypoxia (Fletcher et al., 2004). This is relevant for the current studies in developing chicks, since the embryos experience a period of hypoxia towards the end of incubation prior to hatch, and then are likely to experience further stress after hatch when handling and testing take place. Within the experiments presented here, the comparison of corticosterone levels between chicks is relative as handling and decapitation will raise corticosterone levels to some degree, but
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all chicks were handled in the same way. We hypothesise that it is the acute increase in circulating corticosterone or noradrenaline at the onset of the hypoxic or anoxic insult that leads to memory impairment after hatch. In support of this, we have recently shown that a single injection of corticosterone (0.2 nmol/egg) onto the chorioallantoic membrane at E10 or E14 is sufficient to mimic the adverse effects of hypoxia on memory ability (Rodricks et al., 2006), as does an injection of noradrenaline at E14 (M.E. Gibbs unpublished). The mechanisms of action of acute prenatal hypoxia, corticosterone or noradrenaline which go on to result in poor memory ability after hatch are not currently known and require further investigation. With respect to the cognitive outcome, the important finding of this study is that there are two vulnerable periods around E10 and E14 during development when the hypoxic insult impacts on subsequent memory processing. Hypoxia at E11 and E12 did not have any subsequent effect of memory, which suggests that hypoxia at E10 and E14 are affecting discrete events in brain development. Indeed, two major developmental milestones occur at E10 and E14. At E10 neuronal migration is essentially complete, but axonal outgrowth, dendritic expansion and synapse formation is just beginning (Tsai et al., 1981). The appearance of astroctytes (as opposed to radial glia), does not occur until E13 or E14 (Striedter and Beydler, 1997; Camm et al., 2005). This developmental distinction suggests that although the insult around E10 may be linked to neuronal development, the insult around E14 could affect astrocytic or neuronal development, or a combination of the two. One may argue that hypoxia causes a deficiency in the ability of the chicks to visually discriminate colours, however, the finding that memory deficits induced by hypoxia can be rescued (Camm et al., 2004), demonstrates that the chick’s ability to discriminate between red and blue is not affected, and thus sight is not affected. Hypoxia on E10 resulted in mild impairment of the righting response at 1 and 2 days after hatch, an effect not apparent after hypoxia at E14. In the past, the righting response has been used as a quantitative indicator of the motor ability of newly hatched chicks when embryos were malnourished prior to incubation (Hill, 1993), which may suggest that E10 hypoxia alters neuromotor function. In fact, the unchanged pecking of the test beads in the discriminated bead task suggests that there is not a motor impairment. The test is however quite similar to a tonic immobility test which assesses the defensive behaviour of animals (including chicks) and therefore may be measuring other aspects of behavioural response, such as fear (Jones, 1989). This result requires further investigation. In conclusion, the present study demonstrates that 24 h of hypoxia at E10 in the developing chick results in the same memory impairments as 4 days of hypoxia beginning on E10. At E14, hypoxia for 24 h or anoxia for 5 min, but not hypoxia for 1 h, produces the same cognitive deficits as 4 days of hypoxia. Hypoxia at E10 adversely affects intermediate and thus long-term memory, whereas insults at E14 only affect consolidation into long-term memory. This shows that if the hypoxic insult is of sufficient severity, then the developmental age at the time of the insult determines the nature of the
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