Hypoxia-Induced Dysfunction in Developing Rat Neocortex

Heiko J. Luhmann and Thomas Kral

Institute of Neurophysiology, University of Düsseldorf, D-40001 Dusseldorf, Germany

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Luhmann, Heiko J. and Thomas Kral. Hypoxia-induced dysfunction in developing rat neocortex. J. Neurophysiol. 78: 1212-1221, 1997. Neocortical slices from young [postnatal day (P) 5-8], juvenile (P14-18), and adult (>P28) rats were exposed to long periods of hypoxia. Field potential (FP) responses to orthodromic synaptic stimulation, the extracellular DC potential, and the extracellular Ca2+ concentration ([Ca2+]o] were measured simultaneously in layers II/III of primary somatosensory cortex. Hypoxia caused a 42 and 55% decrease in the FP response in juvenile and adult cortex, respectively. FP responses recorded in slices from young animals were significantly more resistant to oxygen deprivation as compared with the juvenile (P < 0.01) and adult age group (P < 0.001) and declined by only 3% in amplitude. In adult cortex, hypoxia elicited, after 7 ± 4.5 min (mean ± SD), a sudden anoxic depolarization (AD) with an amplitude of 14 ± 6 mV and a duration of 0.89 ± 0.28 min at half-maximal amplitude. Although the AD onset latency was significantly longer in P5-8 (12.5 ± 4.9 min, P < 0.001) and P14-18 (8.7 ± 3.2 min, P < 0.002) cortex, the amplitude and duration of the AD was larger in young (45.7 ± 7.6 mV, 2.19 ± 0.71 min, both P < 0.001) and juvenile animals (29.9 ± 9.1 mV, P < 0.001, 0.96 ± 0.26 min, P > 0.05) when compared with the adults. The hypoxia-induced [Ca2+]o decrease was significantly (P < 0.002) larger in young cortex (1,115 ± 50 µM) as compared with the adult (926 ± 107 µM). Prolongation of hypoxia after AD onset for >5 min elicited in young and juvenile cortex a long-lasting AD with an amplitude of 40.5 mV associated with a decrease in [Ca2+]o by >1 mM. On reoxygenation, only slices from these age groups showed spontaneous repetitive spreading depression in 3 out of 26 cases. In adults, the same protocol caused a significantly (P < 0.05) smaller and shorter AD and never a spreading depression. However, recovery in synaptic transmission after this long-term hypoxia was better in young and juvenile cortex, indicating a prolonged or even irreversible deficiency in synaptic function in mature animals. Application of ketamine caused a 49% reduction in the initial amplitude of the AD in juvenile cortex but did not significantly affect the AD in slices from adult animals. These data indicate that the young and juvenile cortex tolerates much longer periods of oxygen deprivation as compared with the adult, but that a sufficiently long hypoxia causes severe pathophysiological activity in the immature cortex. This enhanced sensitivity of the immature cortex is at least partially mediated by activation of N-methyl-D-aspartate receptors.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

A large number of experimental studies have demonstrated that the immature brain is very resistant to oxygen deprivation. In 1953, Hicks reported in a developmental study on rats and mice that "the newborn animal could resist anoxia for nearly an hour without damage" (Hicks 1953). Subsequent studies in different central nervous structures, such as the brain stem (Ballanyi et al. 1992), the hippocampus (Cherubini et al. 1989; Friedman and Haddad 1993; Kawai et al. 1989), and the cerebral cortex (Bickler et al. 1993; Hansen 1977; Luhmann et al. 1993) confirmed Hicks' results (for review, see Ben-Ari 1992; Haddad and Jiang 1993; Hansen 1985). This striking insensitivity of the immature brain to lack of oxygen results from developmental differences in brain energy production (ATP production via anaerobic glycolysis) and energy consumption (for review, see Ben-Ari 1992; Hansen 1985; Luhmann 1996). Although these experimental observations clearly demonstrate a remarkable resistance of the immature brain to hypoxia, clinical data suggest that oxygen deprivation and ischemia during early ontogenetic development may provoke irreversible structural and functional modifications (for review, see Hill 1991; Latchaw and Truwit 1995; Roland and Hill 1995). Prolonged hypoxia-ischemia during the pre- or perinatal period causes neuronal malformations that may form the structural basis for intellectual and behavioral deficits (Kornhuber et al. 1985) or severe functional disorders (Palmini et al. 1991). Experimental studies may give answers to the questions under what conditions the immature brain develops irreversible deficiencies and which cellular and molecular mechanisms contribute to this process. Recent results from observations on the developing brain suggest that a number of different processes may cause severe disorders during early ontogenesis. gamma -amino-butyric acid (GABA), the main inhibitory transmitter in the adult brain, functions as an excitatory transmitter during early developing in different central structures (for review, see Cherubini et al. 1991; Luhmann and Prince 1992; Sutor and Luhmann 1995). Activation of GABAA receptors in the immature cerebellum (Connor et al. 1987), hippocampus (Segal 1993) and cerebral cortex (Yuste and Katz 1991) leads to activation of voltage-dependent Ca2+ channels and a rise in the intracellular Ca2+ concentration. Under physiological conditions, this mechanism influences gene expression (Bading et al. 1993), growth cone behavior (Kater and Mills 1991; Obrietan and Van den Pol 1996), programmed cell death (for review, see Franklin and Johnson 1992), neuronal migration (Behar et al. 1996; Komuro and Rakic 1992), and cell differentiation (Spitzer et al. 1994) and may represent a prerequisite for the normal maturation of the brain. However, under pathophysiological conditions, such as hypoxia and ischemia, abnormal increases in the intracellular Ca2+ concentration may induce alterations in gene expression (for review, see Schreiber and Baudry 1995) and failures in activity-dependent developmental processes (for review, see Mody and Soltesz 1993). Another factor that strongly influences the normal maturation of different brain regions is the N-methyl-D-aspartate (NMDA) receptor (for review, see Singer 1995). However, enhanced activation of the NMDA receptor under pathophysiological conditions also may induce structural abnormalities, e.g., modifications in neuronal migration, which depends on activation of the NMDA receptor (Komuro and Rakic 1993). These results from experimental studies and the clinical observations raise the following questions: Under what conditions does the immature brain reveal a pathophysiological pattern, which subsequently may induce secondary structural or functional deficits? How does the developing brain differ in its responsiveness to pathophysiological conditions when compared with the adult? Does intracellular Ca2+ elevation or NMDA receptor activation play any role in the pathophysiology of the immature brain under hypoxic conditions? To address these questions, we have chosen the cerebral cortex of young, juvenile, and adult rats and performed extracellular in vitro recordings with ion-sensitive electrodes under normoxic and hypoxic conditions.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Slice preparation

The methods for preparing and maintaining rat neocortical slices in vitro were similar to those described previously (Kral et al. 1993; Luhmann and Heinemann 1992). Young [postnatal day (P) 5-8; day of birth = P0], juvenile (P14-18), and adult (>P28) Wistar rats were anesthetized deeply by hypothermia (P5-8) or intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt) and decapitated. The brain was quickly removed and stored for 1-2 min in ice-cold artificial cerebrospinal fluid (ACSF) consisting of (in mM) 124 NaCl, 3 KCl, 1.25 NaH2PO4, 1.8 MgCl2, 1.6 CaCl2, 26 NaHCO3, and 10 glucose with a pH of 7.4 when saturated with 95% O2-5% CO2. Whole brain coronal slices of a nominal thickness of 500 (P5-8), 450 (P14-18), and 400 (>P28) µm were cut on a Dosaka vibratome (Kyoto/Japan) and trimmed to smaller pieces. Slices prepared from immature animals were cut thicker because in the recording chamber they collapsed to a larger extent in thickness as compared with slices from mature rats. The slice thickness during recording was comparable in all three age groups and amounted to ~350 µm. Slices which according to the criteria of Zilles and Wree (1985) included the primary somatosensory cortex were transferred to an incubation-storage chamber or to an interface-time recording chamber and kept at 32-33 and 34-35°C, respectively. Slices were allowed to recover for >= 1.5 h before recording began.

Extracellular recordings, ion-sensitive electrodes, and induction of hypoxia

Extracellular recordings were performed with 2-5 MOmega electrodes filled with ACSF or with double-barrelled ion-sensitive/reference electrodes manufactured with the Fluka 21048 cocktail (Heinemann et al. 1977). The reference electrode contained 150 mM NaCl. The Ca2+-sensitive electrodes responded to a tenfold change in the extracellular Ca2+ concentration ([Ca2+]o) with a potential shift of 26-30 mV. Extracellular field potential (FP) responses in layers II/III to orthodromic synaptic stimulation of the underlying layer VI were recorded to evaluate the functional status of the slice. Only slices with FP responses of >1 mV in amplitude were selected for further experimental analysis. FP responses were elicited at intervals of 10-30 s to avoid any run-down of synaptic responses especially in immature cortex. Extracellular DC recordings and Ca2+ measurements were displayed on a storage oscilloscope and on a thermo chart recorder (Astromed). Hypoxia was induced in the interface-time recording chamber by aeration with 95% N2-5% CO2. Previous studies have shown that this protocol causes a rapid decline in the tissue O2 tension at the slice surface from ~300 mmHg under normoxia to 0 or near 0 mmHg during hypoxia both in neonates and adults (Jiang et al. 1991), indicating that cortical slices from the three age groups were exposed to the same degree of hypoxia at all depths of the tissue (for a discussion of this issue, see also Nolan and Waldrop 1996). Two different protocols were used to monitor hypoxia-induced dysfunction in developing rat cerebral cortex. In the first set of experiments, hypoxia was terminated at the onset of the sudden anoxic depolarization (AD), and slices were reoxygenated with 95% O2-5% CO2 ("short-term hypoxia"). If no AD occurred, hypoxia usually was terminated after 20 min. This protocol also was used to investigate hypoxia-induced changes in synaptic responses. The FP amplitude was measured under normoxic control conditions and immediately before the AD onset or at the end of the 20-min hypoxia period when no AD occurred. In another experimental protocol, hypoxia was prolonged for >= 5 min after the onset of the AD, and after that period, slices were reoxygenated ("long-term hypoxia").

For pharmacological analyses, the selective and noncompetitive NMDA receptor antagonist ketamine (Sigma, Basel/Switzerland) was bath applied in a concentration of 100 µM >= 30 min before the induction of hypoxia.

Data analysis and statistics

The hypoxia-induced AD was analyzed in its onset latency from the beginning of the N2 aeration, its maximal DC amplitude and maximal [Ca2+]o decrease. In the short-term hypoxia experiments, the DC and Ca2+ signal also was quantified in its duration at half-maximal amplitude. In the long-term hypoxia experiments, the AD amplitude and [Ca2+]o decline was measured at its initial peak and 5 min after the AD onset to determine changes in these parameters under hypoxic conditions. The degree of recovery in synaptic function after an AD of variable duration was estimated by measuring the FP amplitude under normoxic control conditions and 20 min after the AD onset during reoxygenation (%FP recovery = FP amplitude 20 min post AD/FP amplitude control × 100).

The Mann-Whitney U test and the chi 2 were used for statistical analyses. If not otherwise noted, values throughout this report are expressed as mean ± SD.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Short-term hypoxia

The response pattern of the neocortex to transient hypoxia was strongly age dependent. Neocortical slices obtained from young rats differed significantly in a number of parameters from those prepared from juvenile and adult animals. Typical FP responses recorded in a neocortical slice from a P7, P17, and an adult rat under normoxic and hypoxic conditions are illustrated in Fig. 1, A and B, respectively. The average FP amplitude in young cortex decreased from5.4 ± 1.7 mV under normoxia to 5 ± 1.3 mV under hypoxia (-3.1 ± 23.1%, n = 10). In juvenile cortex, the FP response declined from 4.1 ± 1.1 mV to 2.5 ± 16 mV (-41.9 ± 34.3%, n = 13, P < 0.01 vs. young), and in adults, hypoxia caused a reduction in the FP amplitude from 3.2 ± 1.2 mV to 1.5 ± 1.2 mV (-54.6 ± 23.1%, n = 39, P < 0.001 vs. young) (Fig. 1C). These data indicate that slices from young rats tolerate long hypoxic periods without any obvious functional disturbances. In addition, nitrogen aeration for 20 min elicited an AD in only 18 out of 46 (39.1%) slices from young rats, which is a significantly (P < 0.001) smaller proportion as compared with juvenile (35 out of 40; 87.5%) and adult animals (57 out of 63; 90.5%; Fig. 2A). Three slices from young rats even tolerated oxygen deprivation for 60 min without any functional impairment. In addition, in the 18 slices from young rats showing an AD, the onset latency was significantly (12.5 ± 4.9 min, n = 18) longer as compared with the juvenile (8.7 ± 3.2 min, n = 34;P < 0.005) and adult age group (7 ± 4.5 min, n = 57; P < 0.001) (Figs. 2B, 3). These data indicate that the immature cortex maintained normal function during long periods of oxygen deprivation. However, this condition persisted only under hypoxia before the sudden occurrence of an AD. Once the AD was triggered, the expression of pathophysiological signals was much stronger in immature cortex as compared with the adult (Fig. 3). The AD amplitude was significantly larger in young rats (45.7 ± 7.6 mV, n = 8) as compared with juvenile (29.9 ± 9.1 mV, n = 16, P < 0.005) and adult animals (14 ± 6 mV, n = 24, P < 0.001; open circle  in Fig. 2C). A significant difference in this parameter also could be detected between the juvenile and adult age group (P < 0.001). The decrease in [Ca2+]o associated with the AD was significantly larger in young animals (1115 ± 50 µM, n = 8) as compared with the juvenile (771 ± 311 µM, n = 15, P < 0.005) and adult rats (926 ± 107 µM, n = 9, P < 0.005; bullet  in Fig. 2C). Similar age-dependent differences also could be observed in the duration of the AD and Ca2+ signal. The duration of the AD measured at half-maximal amplitude was significantly longer in slices from young animals (2.19 ± 0.71 min, n = 8) when compared with the juvenile (0.96 ± 0.26 min, n = 13, P < 0.001) and adult age group (0.89 ± 0.28 min, n = 24, P < 0.001; open circle  in Fig. 2D). The age-dependent change in the duration of the Ca2+ signal measured at half-maximal amplitude (bullet  in Fig. 2D) was almost identical with the relationship between the AD duration and age.


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FIG. 1. Effects of hypoxia on stimulus-evoked field potential (FP) responses in young (1), juvenile (2), and adult (3) cortex. A: FP responses recorded in layers II/III to electrical stimulation of the afferents in layer VI under normal oxygen supply. B: same as in A, but after 20 min of hypoxia (1) or before the sudden onset of the anoxic depolarization (AD: 2 and 3). C: average percentage decrease in the FP response amplitude during hypoxia in young (n = 10), juvenile (n = 13), and adult (n = 39) cortex. Data are expressed as means ± SE.


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FIG. 2. Properties of the AD in neocortical slices obtained from young (P5-8), juvenile (P14-18), and adult (>P28) rats. In all measurements, hypoxia was terminated at the onset of the AD or continued for a maximal duration of 20 min when an AD could not be elicited (short-term hypoxia). A: percentage of slices from young (n = 46 slices), juvenile (n = 40), and adult (n = 63) rats showing an AD within 20 min of oxygen deprivation. B: average onset latency of the AD in slices obtained from young (n = 18 slices), juvenile (n = 34), and adult (n = 57) rats. C: Maximal AD amplitude (left y axis, open circle ) and extracellular [Ca2+] decrease (right y axis, bullet ) in 3 age groups. D: duration at half-maximal amplitude of the AD (left y axis, open circle ) and the extracellular Ca2+ signal (right y axis, bullet ) in 3 age groups. All data are expressed as means ± SE.


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FIG. 3. Developmental differences in response pattern of the rat neocortex to transient hypoxia under in vitro conditions. Simultaneous recordings of the extracellular DC potential (1) and extracellular Ca2+ concentration (2) were performed in a somatosensory cortical slice from a P7 (A), P17 (B), and adult (C) rat. Duration of hypoxia is given above trace 1 for each age group. Slices were reoxygenated at the onset of the AD (up-arrow  N2 off). Note large amplitude and duration of the AD in the DC recording and Ca2+ signal in the young rat.

The extent of functional recovery after hypoxia and an AD of variable duration was analyzed by measuring FP responses under normoxic control conditions (Fig. 4A) and by comparing these FP amplitudes to the responses obtained 20 min after onset of the AD (Fig. 4B). Plots of these values of recovery in synaptic function against the AD duration at half-maximal amplitude clearly demonstrate an age-dependence in the degree of recovery after hypoxia (Fig. 4C). In slices obtained from adult rats, ADs between 2 and 4 min in duration were associated with no or only partial (<20%) recovery (bullet  in Fig. 4C). In contrast, FPs recorded in slices from young (× in Fig. 4C) and juvenile (open circle  in Fig. 4C) rats recovered after an AD with comparable duration to a much larger extent. These data indicate that the immature cortex is capable of regaining synaptic function after longer periods of oxygen deprivation when compared with the adult. However, the linear regression plots in Fig. 4C suggest that even in the young and juvenile cortex this recovery process is limited to ADs, which do not last longer than ~10 min.


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FIG. 4. Age dependence of recovery in synaptic function following an AD. A: control FP responses under normoxic conditions in young (1), juvenile (2), and adult (3) cortex. B: same as in A, but 20 min after the onset of an AD, which lasted at half-maximal amplitude 4.8 min (1), 3.1 min (2), and 2.6 min (3). C: relationship between AD amplitude at half-maximal amplitude and recovery of FP responses in young (×, n = 7,y = -12x + 121, r2 = 0.5), juvenile (open circle , n = 12, y = -10x + 66,r2 = 0.62), and adult cortex (bullet , n = 18, y = -33.5x + 104, r2 = 0.8).

Long-term hypoxia

In agreement with our observations on hypoxia-induced dysfunction using the short-term hypoxia protocol, longer periods of oxygen deprivation also elicited age-dependent modifications in the extracellular DC potential and Ca2+ signal. Prolongation of hypoxia for >= 5 min after the AD onset elicited an AD and Ca2+ response that was larger and longer in immature cortex as compared with the adult (Fig. 5). The initial AD amplitude measured 10-30 ms after AD onset was significantly smaller in adults (12.2 ± 6.3 mV,n = 32) as compared with the young (40.5 ± 7.4 mV, n = 10, P < 0.001) and juvenile rats (40.5 ± 11.7 mV, n = 16, P < 0.001; Fig. 5 and open circle  in Fig. 6A). A comparable result could be obtained for the hypoxia-induced decrease in [Ca2+]o, which was significantly smaller in adult rats (763 ± 135 µM, n = 9) when compared with the young (1084 ± 84 µM, n = 10, P < 0.001) and juvenile age group (1042 ± 93 µM, n = 14, P < 0.001; Fig. 5 and bullet  in Fig. 6A). A rather unexpected result was the response of the immature cortex to long-term hypoxia. Prolongation of oxygen deprivation for >= 5 min caused a decrease in the amplitude of the AD and Ca2+ signal in the adults but almost no change in the young age group (Fig. 5). Five minutes after the AD onset, the AD amplitude in young and juvenile cortex amounted to 36.7 ± 8.6 mV (n = 10) and 27.2 ± 11.6 mV (n = 15), respectively, whereas in adults the AD decreased to an average amplitude of 6 ± 5.2 mV (n = 28; open circle  in Fig. 6B). These data indicate that in adults the AD and Ca2+ signal decreased in amplitude under hypoxic conditions by 57.4 ± 21.6% (n = 27) and 43.5 ± 9% (n = 8), respectively (Fig. 6C). Such a pronounced recovery could not be observed in young and juvenile animals. In young cortex, the relative decrease in the AD amplitude (9.9 ± 9.5%, n = 10) and Ca2+ response (11.9 ± 7.7%, n = 10) was significantly smaller when compared with the adults (both P < 0.001) (Fig. 6C). Similar results could be obtained in juvenile animals, which showed a recovery in the AD amplitude by 32.1 ± 16.8% (n = 11, P < 0.002 vs. adult) and in theCa2+ signal by 16 ± 9.9% (n = 10, P < 0.001 vs. adult; Fig. 6C).


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FIG. 5. Simultaneous extracellular DC recordings (1) and extracellular [Ca2+] measurements (2) in a neocortical slice from a P8 (A), P17 (B), and an adult rat (C). Hypoxia was prolonged for >5 min after the onset of the AD to monitor changes under long-term oxygen deprivation. The duration of hypoxia preceding the AD is indicated above trace 1 for each age group (down-arrow ). Slices were reoxygenated at the time point marked by up-arrow .


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FIG. 6. Age-dependent properties of the AD during long-term hypoxia. In all measurements, oxygen deprivation was prolonged for >5 min after the onset of the AD to monitor changes in the AD amplitude and extracellular calcium signal under hypoxia. A: initial maximal DC amplitude of the AD (left y axis, open circle ) and maximal extracellular [Ca2+] decrease (right y axis, bullet ) measured immediately after the AD onset. B: AD amplitude (left y axis, open circle ) and extracellular [Ca2+] decrease (right y axis, bullet ]) determined under hypoxic conditions 5 min after the AD onset. C: percentage decrease of the AD amplitude (left y axis, open circle ) and percentage recovery of the extracellular Ca2+ signal (right y axis, bullet ) within 5 min after the AD onset. Data were obtained in neocortical slices from young (n = 10 slices), juvenile (n = 16), and adult (n = 32) rats and are expressed as means ± SE.

Beside these pronounced developmental differences in the responsiveness to hypoxia, neocortical slices from immature and adult rats also differed in the expression of spontaneous pathophysiological activity after long-term hypoxia. Two slices from young animals (n = 10) and one slice from a juvenile rat (n = 16) showed, during the reoxygenation period after prolonged hypoxia, three to five spreading depression episodes, which varied in amplitude from 35 to 50 mV and in duration at half-maximal amplitude from 10 to 50 s (Fig. 7A). Each spreading depression episode was accompanied by a simultaneous decrease in [Ca2+]o to 100-150 µM (Fig. 7B). Although spreading depression could be elicited easily and reliably in adult cortex under normoxic conditions by local application of high K+ (Krüger et al. 1996), we never observed a spreading depression in adult cortex during or after prolonged hypoxia (n = 32). Although posthypoxic spontaneous spreading depression was observed in only 3 out of 26 slices of immature rats, this result was significant when compared with the adults (0 out of 32; P < 0.05). These data indicate that immature cortical slices have a lower threshold to generate spreading depression after prolonged oxygen deprivation as compared with mature neocortex.


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FIG. 7. Repetitive spreading depression recorded in a P16 rat neocortical slice after long-term hypoxia (20.5 min). After 14.5 min hypoxia, a prominent AD could be monitored simultaneously in the extracellular DC recording (A) and the Ca2+ signal (B). After prolongation of hypoxia for another 6 min, the slice was reoxygenated (up-arrow  N2 off). During the recovery period, 5 spontaneous spreading depression episodes could be observed in the DC recording and the calcium signal.

Effects of ketamine

To investigate the role of NMDA receptors in the enhanced sensitivity of the immature cortex to hypoxia, we analyzed the influence of the noncompetitive NMDA antagonist ketamine on cortical dysfunction during long-term hypoxia. Ketamine was bath applied in a concentration of 100 µM >= 30 min before oxygen deprivation. These experiments were limited to juvenile and adult rats because the probability to evoke an AD was significantly higher in the former age group as compared with the young animals (Fig. 2A). The AD onset latency in juvenile cortex was 10.03 ± 2.12 min (n = 9), indicating that ketamine caused a small but insignificant delay in the AD onset by 1.33 ± 2.12 min when compared with the data obtained in normal ACSF (Fig. 8A). In adults, the AD occurred at a latency of 9.77 ± 7.7 min (n = 13), suggesting that in this age group ketamine application induced an increase in the AD onset by 2.81 ± 7.7 min (n = 13; Fig. 8A). However, this effect was also not significant at the P < 0.05 level. A prominent ketamine effect could be observed in juvenile rats on the amplitude of the AD. In this age group, the initial AD amplitude amounted to only 20.7 ± 3.1 mV (n = 9), suggesting that ketamine caused a significant (P < 0.001) reduction in this parameter by 19.8 ± 3.1 mV (n = 9) when compared with the measurements in normal ACSF (Fig. 8B). In addition, [Ca2+]o decreased by only 856 ± 83 µM (n = 9), indicating that ketamine induced a significant (P < 0.002) reduction in the initial Ca2+ signal by 186 ± 83 µM (n = 9) when compared with the control recordings (Fig. 8B). In adult cortical slices, ketamine did not significantly change the initial AD parameters (Fig. 8B). The AD amplitude was 13.5 ± 8.1 mV (n = 22) and [Ca2+]o decreased by 609 ± 262 µM (n = 17). When compared with the control recordings in normal ACSF, ketamine caused an increase in the AD amplitude by 1.3 ± 8.1 mV and a reduction in the Ca2+ signal by 153 ± 262 µM (both P > 0.05; Fig. 8B). These data suggest that in contrast to the adult cortex, ketamine profoundly influenced the responsiveness of the juvenile cortex to hypoxia.


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FIG. 8. Effects of the N-methyl-D-aspartate antagonist ketamine (100 µM in bath) on the AD properties in slices obtained from juvenile (n = 9 slices) and adult (n = 22) rats during long-term hypoxia. A: average differences in the AD onset latency in slices exposed to ketamine as compared with measurements in normal artificial cerebrospinal fluid. B: differences in the initial AD amplitude (left y axis, square ) and initial extracellular Ca2+ signal (right y axis, black-square) measured in ketamine-exposed slices as compared with control data. C: same as in B, but measurements were obtained 5 min after the AD onset. Note much larger effects of ketamine in the juvenile age group as compared with the adults. Data are expressed as means ± SE.

A prominent ketamine effect also could be observed in juvenile cortex during the late hypoxia period. Five minutes after the onset of the AD, the AD amplitude and [Ca2+]o decrease amounted to only 8.9 ± 3.3 mV (n = 9) and 623 ± 207 µM (n = 8), respectively. These data indicate that ketamine caused a significant reduction in the AD amplitude and the Ca2+ signal by 18.3 ± 3.3 mV (n = 9, P < 0.001) and 352 ± 207 µM (n = 8, P < 0.002), respectively (Fig. 8C), when compared with the controls. In adults, the AD amplitude in ketamine amounted after 5 min hypoxia to 6.5 ± 5.6 mV (n = 22), which was 0.5 ± 5.6 mV (n = 22) larger as compared with the result obtained in normal ACSF (P > 0.05, Fig. 8C). In contrast, the decrease in [Ca2+]o measured 5 min after AD onset was 419 ± 198 µM (n = 17) in ketamine and therefore 259 ± 198 µM (n = 17) smaller as compared with the adult controls (P < 0.005, Fig. 8C).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Hypoxia-induced dysfunction in developing cortex

Neocortical slices from young animals could tolerate long periods of oxygen deprivation without any obvious functional deficit. In 61% of the neocortical slices from P5-8 rats, synaptic transmission was well preserved under hypoxia and the extracellular DC potential and Ca2+ concentration was stable for 20 min or longer. Young and juvenile cortex also showed a notable recovery in synaptic function following an AD. In contrast, neocortical slices from adult rats were much more sensitive to hypoxia and showed a marked suppression in synaptic responsiveness, no or only minimal recovery in synaptic transmission after an AD, and an ~90% incidence of expressing an AD. These data are in good agreement with a large number of previous reports on the sensitivity of the immature brain to hypoxia and ischemia (for review, see Ben-Ari 1992; Haddad and Jiang 1993; Hansen 1985). A prominent insensitivity to oxygen deprivation has been described in CA1 hippocampal neurons from P1-4 rats in vitro (Cherubini et al. 1989), in P4/P7 rat cerebral cortex in vivo (Hansen 1977), in P5-8 rat somatosensory cortex in vitro (Luhmann et al. 1993), and in dissociated CA1 neurons from P1-8 rats (Friedman and Haddad 1993). The relative resistance of the immature cortex to hypoxia probably results from a low metabolic activity, a greater ATP reserve (Kawai et al. 1989), the capacity of anaerobic glycolysis (Bickler et al. 1993), a reduced oxygen free radical production (Schreiber et al. 1995), and, under in vivo conditions from age-dependent changes in vascular supply, cerebral blood flow responsiveness and oxygen-binding capacity of blood (Grafe 1994). Although these data clearly indicate that the immature cortex is relatively insensitive to hypoxia, our present data suggest that a sufficiently long period of oxygen deprivation causes pronounced pathophysiological activity in young animals. Both, the amplitude of the hypoxia-induced extracellular DC deflection and the Ca2+ signal was significantly larger in immature cortex as compared with the adult. In young cortex, both signals were also significantly longer in duration when compared with the older age groups. Similar results have been reported by Yager et al. (1996) using a physiologically controlled in vivo model of hemispheric global ischemia. Brain damage was most severe in 1- and 3-wk-old rats, followed by 6-mo-old animals, and significantly less in the 6- and 9-wk-old group. These data indicate that the age-dependent expression of ischemia-induced brain damage shows a U-shaped form with a peak in very early and late postnatal life (see Fig. 2 in Yager et al. 1996). Our observations also are supported by Friedman and Haddad (1993), who measured with fluo-3 the intracellular Ca2+ concentration in freshly dissociated CA1 neurons from neonatal (P1-8) and adult (P21-40) rats. With an average latency of 1.7 min in adults and 8.9 min in neonates, anoxia caused a significant increase in fluorescence, which only in P1-8 neurons continued to increase during reoxygenation (Friedman and Haddad 1993). These results correlate well with our observations in immature cortex, indicating that a similar process occurs in isolated cells and brain slices.

Our data on age-dependent changes in the AD duration are also in agreement with in vitro observations on the properties of 4-aminopyridine-induced spreading depression (SD) episodes in developing rat hippocampus (Psarropoulou and Avoli 1993). The SD, an AD-like phenomena, was significantly longer in P2-10 animals (169 s) as compared with the P21-30 age group (55.5 s). These developmental differences in the duration of the AD or SD may reflect the gradual maturation of ionic pump mechanisms, such as the Na+, K+-ATPase (Fukuda and Prince 1992), both in neurons and in glial cells. The development of glial cells, especially astrocytes, may explain some of our observations in immature cortex under hypoxic conditions. Astrocytes in rat cerebral cortex develop gradually within the first seven postnatal weeks (Stichel et al. 1991). Because glial cells possess a large number of ion channels and transport mechanisms, they profoundly contribute to ion fluxes and play a key role in hypoxic-ischemic dysfunction (for review, see Walz et al. 1993). Furthermore, the maturation of glial cells parallels the decrease of the extracellular space (ECS) during ontogenesis (Lehmenkühler et al. 1993). The ECS volume fraction alpha  (the relative tissue volume available for diffusion of extracellular substances) measured in layer III of the rat somatosensory cortex decreases significantly from 32% at P4-5 to 22% at P20-21 (Lehmenkühler et al. 1993). In contrast, the ECS tortuosity lambda  (increased average path length for diffusion of particles between two points due to barriers from cellular membranes etc.) and the nonspecific, concentration-dependent cellular uptake remain relatively constant (Lehmenkühler et al. 1993). These data indicate that during postnatal development, the ECS in the rat cortex shrinks by ~30% because of the maturation of the glial cell syncytium. This age-dependent decrease in the ECS is associated with a relative increase in the resistivity of the interstitium in adult cortex even under physiological conditions. Under the assumption that the hypoxia-induced shrinkage of the ECS in the young cortex is the same as in the adult (~70%) (see Jing et al. 1994), then the ECS would amount to 9.6% in P4-5 cortex and 6.6% in P20-21 cortex during hypoxia. These values are in the range of observations by Pérez-Pinzón et al. (1995) in rat neocortex in vitro(9%) and data by Syková et al. (1994) in rat spinal cordin vivo (5%).

Age-dependent changes in the ECS may explain partly our result on the larger Ca2+ signal in immature cortex because a given number of extracellular Ca2+ ions would dilute to a lower concentration in a larger ECS in the young cortex. However, on the basis of these data, one also would expect that the AD amplitude should be larger in adults because extracellular K+ and excitatory amino acids accumulate to a greater extent in a reduced ECS, thereby causing a larger cellular depolarization. Other age-dependent processes, such as voltage- and transmitter-activated Ca2+ channels, membrane leakage, and reversed Na+-Ca2+ exchange (for review, see Waxman et al. 1991), also may influence the larger responses of the immature cortex to hypoxia. Because Ca2+-binding proteins generally appear after neurons have begun to differentiate (for review, see Baimbridge et al. 1992), intracellular Ca2+ buffering may be less functioning in the young brain as compared with the adult. Furthermore, the depolarization- and Ca2+-induced release of Ca2+ from intracellular stores is highest at early stages of development and declines during further development (Kocsis et al. 1994). All these mechanisms contribute to a larger intracellular Ca2+ concentration in the immature brain, which under physiological conditions may be necessary for the normal ontogenesis (see INTRODUCTION). However, under pathophysiological conditions, increased and prolonged intracellular Ca2+ levels may induce irreversible modifications in the developing brain.

Another interesting outcome of the present study is the observation that hypoxia lowers the threshold for the generation of SD during the reoxygenation period in immature cortex. Only in young and juvenile cortical slices, repetitive spontaneous SD episodes could be observed during the early reoxygenation period after prolonged hypoxia. Similar SD-like events have been described previously in rat cortex in vivo after occlusion of the middle cerebral artery (Iijima et al. 1992). P5-17 rats also showed acute electrocortical seizure activity when exposed to transient hypoxia (Jensen et al. 1991), and oxygen deprivation at this age significantly lowered the threshold for the manifestation of epileptic seizures in adults (Romijn et al. 1994). These data suggest that the sensitivity of the cortex to hypoxia shows a transient peak during early ontogenetic development. Because this peak in rat cortical development coincides with the maturational status of the perinatal human cerebral cortex (Romijn et al. 1991), these results may explain why severe hypoxia-ischemia during the perinatal period produces irreversible structural damage also in the human cortex (Finer et al. 1981; for review, see Hill 1991).

Role of NMDA receptors in pathophysiological activity during early development

NMDA receptors play an important role in neuronal plasticity during early cortical development (for review, see Singer 1995). Experimental and clinical data strongly indicate that NMDA receptors also are involved in the generation of different pathological and pathophysiological conditions (for review, see Choi 1992; Coyle and Puttfarcken 1993). Our observations suggest that NMDA receptors play a major role in hypoxia-induced dysfunction during early cortical development. In agreement with a previous report (Kral et al. 1993), NMDA receptor blockade delayed the AD onset. This effect was slightly larger in adults as compared with the juvenile age group. The initial AD amplitude and the amplitude of the AD after prolongation of hypoxia for 5 min was significantly larger in the immature cortex as compared with the adult. In juvenile cortex, the noncompetitive NMDA antagonist ketamine reduced both parameters significantly by 49 and 67%, respectively, whereas in adult cortex, both parameters increased in ketamine insignificantly by 11 and 8.5%, respectively. These data suggest that NMDA receptors contribute to the AD amplitude only in juvenile cortex. In contrast to the ketamine effect on the initial and late AD amplitude, the influence of ketamine on the hypoxia-induced [Ca2+]o decrease was not age dependent. In both age groups, ketamine reduced the early Ca2+ signal by 18-20% and the late [Ca2+]o decline by 36-38%, indicating that the Ca2+ influx via the NMDA receptor was not significantly different between these age groups. Our data indicate that the larger AD amplitude in immature cortex is mediated substantially by ionic fluxes other than Ca2+ influx. Therefore, NMDA receptor-mediated Na+ influx and K+ efflux may be enhanced under hypoxic conditions in young cortex as compared with the adult.

The dominant role of the NMDA receptor in mediating hypoxia-induced dysfunction in immature cortex is in good agreement with Fura-2 measurements in developing rat cortex by Bickler et al. (1993). The noncompetitive NMDA antagonist MK-801 (150 µM) reduced the hypoxia-induced intracellular Ca2+ elevation significantly only in 7- and 8-day-old rats, but not in older animals. This age-dependence may result from developmental changes in the NMDA receptor subunit composition (Monyer et al. 1994; Sheng et al. 1994; Williams et al. 1993), its glycine (Kleckner and Dingledine 1991) or magnesium sensitivity (Ben-Ari et al. 1988; Burgard and Hablitz 1994; Kleckner and Dingledine 1991; Morrisett et al. 1990), a transient increase in NMDA receptor density (Bode-Greuel and Singer 1989; Insel et al. 1990), or ontogenetic changes in the redox modulation of the NMDA receptor. In addition, in adult neurons Ca2+ influx through NMDA channels activates the Ca2+/calmodulin-dependent phosphatase 2B (calcineurin), which shortens the duration of NMDA channel openings (Lieberman and Mody 1994). Because calcineurin occurrs gradually after P4 (Polli et al. 1991), this Ca2+-dependent negative feedback control of the NMDA receptor is less effective in immature neurons as compared with the adults. All these factors contribute to a transient NMDA receptor-mediated hyperexcitability during early cortical development (Luhmann and Prince 1990). Whereas under physiological conditions this enhanced NMDA receptor activation may be a prerequisite for the normal maturation of the cortical network (Kleinschmidt et al. 1987; Komuro and Rakic 1993), overstimulation of NMDA receptors under pathophysiological conditions may cause acute dysfunction and long-term structural damage (Ikonomidou et al. 1989; Luhmann et al. 1995; Urban et al. 1990; for review, see Choi 1992; Coyle and Puttfarcken 1993). These data also suggest that prevention of hypoxia- and ischemia-induced injury by NMDA-receptor blockade may be more beneficial in immature brain as compared with the adult (Ford et al. 1989; Hattori et al. 1989; Olney et al. 1989).

    ACKNOWLEDGEMENTS

  This work was supported by grants SFB 194-B4 and Lu 375/3-1 from the Deutsche Forschungsgemeinschaft to H. J. Luhmann.

    FOOTNOTES

   Present address of T. Kral: Dept. of Neurosurgery, University of Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany.

  Address for reprint requests: H. J. Luhmann, Institute of Neurophysiology, University of Dusseldorf, PO Box 101007, D-40001 Dusseldorf, Germany.

  Received 22 October 1996; accepted in final form 29 May 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society