Hypoxia-induced hyperexcitability in vivo and in vitro in the immature hippocampus

EPILEPSY RE.ARCH ELSEVIER

Epilepsy Research 26 {1996) 13i - t 40

Hypoxia-induced hyperexcitabflity in vivo and in vitro in the immature hippocampus Frances E. Jensen

Carl Wang

a Department t~fNeurology. Children's Hospital. Han'ard Medical School Boston. MA 021 I5. USA b Program in N-,uroscience. Harcard Medical School. Boston. MA 02115. USA

Received 7 November 1995: accepted 7 November 1995

Abstract Hypoxia is the most common cause of neonatal seizures and encephalopathy. We have previously developed an in vivo experimental model of perinatal hypoxia which exhibits age-dependent acute and chronic epileptogerdc effects. Between postnatal day (P) 10-12, the rat exhibits acute seizure activity during global hypoxia, while no seizures are induced at earlier (P5) or older (P60) ages. Rats exposed to hypoxia between PI0-12 have reduced seizure thresholds to chemical convulsants in adulthood. The nonNMDA antagonist NBQX appears to suppress both the acute and long term epileptogenic effects of hypoxia. The agz-dependency of the hyperexcitable response to hypoxia in vivo can be reproduced in vitro using hippocampal slices. In Mg2+-free media, hypoxia induced ictal discharges within 60 s of onset in 79% of slices from normal P I0 rat pups compared to 11% of adult slices (p < 0.01 ). Model systems such as that described here allow for correlation of in vitro and in vivo electrophysiology and should previde data regarding the pharmacological and physiological characteristics of hypoxia-induced seizure activity in the immature brain which could ultimately be applied to therapeutic strategies. Keywords:

Epilepsy; Asphyxia; Temporal lobe epilepsy; Neonatal; Glutamate; Ischemia

1. I n t r o d u c t i o n Hypoxia is the most c o m m o n cause of neonatal seizures and encephalopathy. Seizures complicating neonatal hypoxia can be prolonged and at times refractory to conventional anticonvulsant therapy. Neonatal hypoxia is a risk factor for the later development o f epilepsy. Clinical experience would suggest that the acute and chronic epileptogenic effects of hypoxia might be age dependent, with the immature brain more susceptible than the adult. The fre-

* Corresponding author. Tel.: +1 (617) 355-8439; fax: +! (617) 738-1542; e-mail: [email protected] 0920-1211/96/$15.00 Published by Elsevier Science B.V. PII S0920-1211(96)00049-6

quency of seizures with onset in the first month of life is higher than at any other time in childhood [1,2]. While seizures are a frequent accompaniment of neonatal hypoxia or hypoxia/ischemia, seizures are relatively u n c o m m o n in adults presenting with h y p o x i a / i s c h e m i a . Furthermore, hypoxia-induced neonatal seizures can be refractory to medical therapy [3,4] and some infants develop chronic epilepsy after recovery from the encephalopathy [5,6]. Perinatal asphyxia resulting in hypoxic-ischemic encephalopathy accounts for 3 0 - 6 5 % of neonatal seizures, with an incidence of approximately 3 per 1000 newborns [3,5,7,8]. Postnatal epilepsy is a frequent complication of neonatal seizures in general,

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F.E. Jensen, C Wang~Epilepsy Research 26 (1996) 131-140

occurring in 20-50% of cases, and often in association with cerebral palsy and mental retardation [9,10]. The relationship between the hypoxic insult and later epilepsy is difficult to resolve from clinical studies. Experimental animal models are needed to determine age-dependent responses to hypoxia which may have an epileptogenic effect. Pathology in the hippocampus is frequently observed in human and animal models of the epilep-

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Fig. 2. Seizure severity during hypoxia at PIO affects seizure susceptibility outcome. Compared to normal control rats (Control), rats which had undergone hypoxia at PI0 (Hypoxic) have reduced thresholds to flurothyl latencies in adulthood. However, if rats that did not exhibit tonic clonic activity are analyzed alone (Hypoxic, - szrk there is no change in seizure susceptibility. In contrast, rats that exhibited tonic clonic seizures during hypoxia at PI0 (Hypoxic, + szr) have significantly lower thresholds than control rats. (From Jensen et al. [15].)

sies. Hippocampal, or mesia! temporal, sclerosis is frequently observed in biopsy and postmortem samples from human temporal lobe epilepsy patients. Seizures during early life and infancy may increase the risk of this form of epilepsy (for review, see Ref.

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Fig. 1. EEG activity during exposure to 3% O 2 in representative animals from each age group. Age determines the degree of epileptiform activity induced during global hypoxia. (A) Spike discharges are seen in isolation, and in association with a myoclonic jerk (MJ) in this rat at postnatal days (P) 5 to 7. (B) Tracing from a PI0 to 12 rat showing train of spike discharges in association with tonic-clonic head and trunk activity (T/C). (C) Rhythmic spike discharges are seen in this rat from the P15 to 17 group, without any associated behavioral changes. (D) Low-amplitude spike acivity seen in this rat from the P25 to 27 group, without any associated behavioral correlation. (E) An adult (P5060) rat exhibits isoelectric EEG during 02 deprivation. For each age, two channels of EEG activity are displayed. ECG activity is shown below the EEG tracings. See methods for details of electrode placement. Movement artifact is labeled where present in both EEG and ECG tracings. (From Jensen et al. [12].)

F.E. Jee~sen, C. WaHg/ ~:pilepsy Research 26 ¢] 9 9 6 ) ]21 ]40

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exhibits both t~e acute a~d chro.~ic epitep~.oge~ic effects of hypoxia. Using this h~ vivo model, we b.ave show~ that a ~5-20 mi~ exposure to moderate gtoba] hypoxia (3-4e~ O,) i~d~ces seizure activity in immature rat pups aged postnatal day (P) 10-12, but not at younger (P5) or older (P60) ages [12] (Fig. l). Furthermore, rats exposed to hypoxia at PlO, b,~t not at you,g,., or . . . . r ,,s,.s, exhibit ;. . . . . . . . a • susceptibility in adulthood
We have previously developed an in vivo experimental model of perinatat hypc.da which exhibits acute and chronic age-dependent epi~eptogenic effects. Here we review data from our previous work, and also report new data indicating that the age-dependent epi|eptogenic effect of hjpoxia can be reproduced in vitro in hippocampal slices.

2, An an~maB rno~e~ o f the acute and c h r o N c epilep~ogen~c effects o f p e r ~ a ~ a l h y p o ~ a

To study these important questions, we have developed an animal model of perinatal hypoxia which

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Fig. 3. The relative effects of the agents administered as treatment prior to hypoxia in the Pl0 ral. (a) Tonic/clonic seizure activily in rats treated with normal saline (NS, control). (b) Lorazepam (1 mg/kg) suppressed tonic clonie seizures in most cases, but myuclonic jerks persisted. In contrast, beth MK-801 (! mg/kg) (c) and NBQX (20 mg/kg)(d) significantly suppressed hypoxia-induced seizure activity.

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F.E. Jensen, C. Wang / Epilepsy Research 26 (1996) 131-140

ited only myoclonic jerks during hypoxia [14,15] (Fig. 2). Routine histology shows that this moderate hypoxia does not result in neuronal death in supratentorial brain regions. Depth electrode recordings and immunoreactivity to the protein product of the immediate early gene c-fos suggest that neocortical and hippocampal structures are predominantly involved in hypoxia-induced seizure activity [ 16,17]. In addition to the age and regional specificity of the response to hypoxia, we have also demonstrated pharmacological specificity. Because glutamate is the major excitatory amino acid (EAA) transmitter in the brain, we have compared the effects of EAA antagonists with those conventional anticonvulsants which are known to facilitate inhibition. Glutamate receptors are involved in physiologic excitatory processes such as neuronal development [18-21], learning and memory [22,23], and synaptic plasticity [22,24]. These receptors, especially the NMDA subtype, also play a critical role in pathologic neuronal events such as hypoxic/ischemic neuronal injury, epilepsy, and certain chronic neurodegenerative disorders [25]. EAA's play important roles in the initiation, spread, and maintenance of seizure activity (for review, see Meldrum [26]). A number of subtypes of ionotropic and metabotropic glutamate receptors exist (for review, see Re(. [27]). In particular, the N-metl',:l-D-aspartate (NMDA) and the ot-amino-3hydr,Jxy-5-methyl-4-isoxazole propionic acid (AMPA) can induce excitotoxic neuronal death in vivo and in vitro (for review, see Refs. [28-31]). There is much evidence to suggest that excessive release of EAA neurotransmitters mediates neuronal damage in the setting of hypoxia/ischemia [25,32]. EAA receptor activation has been implicated in seizures [33], and EAA's are elevated in epileptic foci of humans [34]. In experimental models, NMDA and nonNMDA antagonists are neuroprotective (for review, see Refs. [35,36]) and anticonvulsant (for review, see Refs. [33,37]). We compared the ability t)i' the NMDA antagonist MK-801, the AMPA antagonist NBQX, and the benzodiazepine Iorazepam to suppress the acute and long term effects of perinatal hypoxia in our rodent model. Pretreatment of P10 rats with NBQX (20 mg/kg), an antagonist at the AMPA receptor subtype, significantly suppressed the acute epileptic response during hypoxia (Fig. 3) and prevented later

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Fig. 4. Latencies to flurothyl-induced myoclonic jerk in P75 rats from different experimental groups. Vehicle contrel rats exposed to hypoxia at PI0 but treated with normal saline (vehicle) had significantly lower latencies than control litter mates, as shown in Fig. 2. NBQX treatment during hypoxia at PIO significantly increased flurothyl latencies compared to hypoxia vehicle controls, whereas treatment with MK-801 or lorazepam did not differ significantly from treatment with vehicle. (From Jensen et al.

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increases in seizure susceptibility in adulthood (Fig. 4). In contrast, neither pretreatment at PI0 with MK-801, an antagonist of the N-methyl-D-aspartate (NMDA) EAA receptor subtype, nor the conventional anticonvulsant lorazepam had significant acute or chronic protective effects [15]. These results suggest that excess AMPA receptor activation may be a feature of hypoxia seizures in the neonatal brain. The lack of effect of the benzodiazepine lorazepam is consistent with its variable effect on these seizures clinically. This whole animal model reproduces many characteristics of perinatal hypoxia in the human. Of note is that the age window between P10-12 approximately corresponds to the term human neonate by biochemical, electrophysiological, and metabolic parameters [38-40]. Our results suggest that a brief hypoxic insult can have long lasting epileptogenic effects, and that these may be mediated by enhanced nonNMDA neurotransmission. These effects are specific to the age window around P10, and hypoxia at

~35

F.E. Jensen, C Wang / Epilep,vy Research 26 ¢1996) 13 / - 140

younger or older ages does not result in enhanced excitability.

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3. In vitro evidence of ageodependen~ effects ~f perinatal hypoxia

H~poxia

Future studies determining the mechanism of this age-dependent epileptogenic effect of hypoxia will require greater control over the experimental conditions than can be achieved in vivo. The in vivo data suggest that hypoxia-induced seizures can be generalized, and little is known about the susceptibility of the hippocampus in particular to hypoxia-induced seizures. Recent experiments were conducted to determine whether the hippocampus in vitro displayed intrinsic age dependent differences in susceptibility to hypoxia-induced seizures. Hippoeampal slices were prepared from P10 rat pups and incubated for l h in control artificial cerebrospinal fluid (ACSF in raM: 124 NaCI, 5 KC1, 1.25 NaHzPO 4, 24 NaHCO s, 0 or 1.5 MgCI~_, 1.8 CaCI,, and 10 o-glucose, at pH 7.4) in an interface slice ciiamber bubbled with 95%Oz/5%CO2 at 37°C. In order to increase excitability of slices from both age groups, the medium was exchanged with Mg 2+free medium for at least 30 min prior to hypoxia and for the remainder of the experiment. Continuous extraceilular recordings were obtained from area CA 1 during the 10 min prior to introduction of MgZ+-free medium and throughout the remainder of the experiment. Recordings were made using glass microelectrodes (1-2 M ~ ) placed 100 /~m below the surface of the slice in s. pyramidale of area CA 1. Continuous traces were acquired using the Neuropro software (R.C. Electronics, pre-publication edition kindly made available by G. Rose, University of Utah). Hypoxia was simulated by interrupting O 2 flow for 60 s as per Kawasaki et al. [41]. Following hypoxia, the chamber and slice fluid were again bubbled with 95%O2/5%CO

2.

Under Mg 2+-free conditions, baseline activity appeared age dependent. In the adult slices (n = 9), Mg2+-free medium induced interictal bursts without ictal discharges (Fig. 5). In contrast, all pup slices (n = 14) responded to Mg2+-free conditions with an initial period of ictal bursting, which would remit within 30-60 min and convert to a pattern of interic-

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tal bursting (Fig. 6). Exposure to hypoxia in vitro also resulted in age-dependent differences in response. Hypoxia did not alter or induced a slow decline in Mg2+-free medium-induced interictal activity to isoelectric state within 60 s in 8 / 9 adult slices (Fig. 5). Immature pup slices were exposed to hypoxia only after at least l0 min of stable interictal activity without any electre~aphic seizures. In 11/14 slices, hypoxia induced ictal seizures within 28 + 3.5 s of its onset, with the average duration of ictal activity being 20.4 __ 2.1 s (Fig. 6). The incidence of ictal activity was significantly greater in the immature slices than in the adult slices, where only 1 / 9 slices exhibited hypoxia-induced ictal activity ( p < 0.01). Reconstitution of normal oxygenated medium resulted in recovery to baseline interictal activity. Because basal temperature is lower in P10 rats than in adult rats (approximately 33°C compared to 37°C), the effect of temperature on the apparent

136

F.E, Jensen, C Wang~Epilepsy Research 26 (1996) 131-140

pocampal pyramidal cells which enhance hypoxia-induced hyperexcitability.

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4. Maturational changes coincidem with window of vulnerability to hypoxia-induced seizures

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age-dependency was controlled for by a second set of experiments in which P10 hippocampal slices were exposed to hypoxia in Mg2+-free medium at 33°C. At 33°C, 12/17 slices exhibited seizures when 02 was interrupted, with an average latency of 32.4 + 3 s, and duration of 16 + 1.6 s. These experiments suggest that the epileptogenic effect of hypoxia in immature hippocampal slices is unlikely to be strongly temperature dependent as seizure incidence, latency to onset, and duration were not significantly different in the pup slices between 37°C and 33°C. Taken together these results suggest that the seizure threshold to hypoxia-induced seizures in Mg2+-free medium is dependent on age, with the immature hippocampus exhibiting a significantly lower threshold to such activity in vitro. Furthermore, these results support the hypothesis that there may be intrinsic synaptic or nonsynaptic properties within specific neuronal subpopulations such as hip-

Hypoxia induced hyperexcitability can be demonstrated in vivo and in vitro, and appears to be highly age-dependent. Modulation of neuronal excitability is dependent on a number of factors which are known to undergo changes during development. Below we consider maturational phenomena which might underlie the age-dependent susceptibility of the brain to epileptogenesis when provoked by a hypoxic insult. The second postnatal week of life in the rat is considered to be analogous to a human infant at term according to parameters of biochemistry, myelination, and electrophysiology [39,40,42,43]. This is also a window of development where there is abundant neuronal growth, synaptogenesis, and evidence of neuronal plasticity. Specifically, we describe maturational changes in neurotransmission, ion homeostasis and energy metabolism occurring during a window of vulnerability to hypoxia-induced seizures. Excitatory amino acid (EAA) neurotransmitters are required for most excitatory actions of the brain. The predominant EAA in the brain is glutamate, and neurotransmission is mediated via several glutamate receptor subtypes (for review, see Ref. [27]). Striking maturational changes in EAA receptor function and gene expression occur during the early postnatal period coincident with heightened susceptibility to hypoxic seizures. The second postnatal week in the rat appears to be a maturational stage in which excitatory mechanisms predominate over those which are inhibitory. "~-~ • , , c maturational cur;e for densities of the different EAA receptors exhibits a transient overshoot in density for both the N-methyl-n-aspartate (NMDA) and the cz-amino-3-hydroxy-5-methyl4-isoxazole propionic acid (AMPA) receptor subtypes. For both receptor subtypes, this overshoot occurs around the second postnatal week in the rat [30,44]. This corresponds to the window of development in the rat where there is suprasensitivity to neuronal injury induced by NMDA (postnatal day

F.E. J~'nsen. C. Wang / Epilepsy Research 26 ~19~6~ / 3 / - ]40

(P)4-14) [30~45] zmd AMPA agonists (Pl0) [4'5]. The immature brain is also more susceptible to seizures induced by NMDA and AMPA than the adult [30,46,47]. EAA's have been shown to have trophic influences on neurons during development [18-21] and regulate activity dependent growth [481. NMDA receptors are also implicated in experiencedependent plasticity in the immature brain, and this plasticity can be blocked with NMDA receptor antagonists such as MK-801 [24,49]. Receptor subunit composition varies with maturation. NMDAR2C [50] and NMDA2RD [51] mRNA expression is transiently elevated in hippocampus during the first 2 postnatal weeks [50]. This presence of NMDAR2C is associated with a decrease in Mg -'+ sensitivity compared to the adult [52-54], and hence its presence would contribute to increased excitability. The AMPA receptor subunits also appeal" to undergo dramatic changes in the first 2 postnatal weeks. Sev?ral studies document an age-dependent alteration in the ratio of GluR1 + 3 / G l u R 2 mRNA in neocortex and hippocampus during this time window [55,56]. Given that t::e presence of GluR2 confers Ca a+ impermeability, this predicts an increase in Ca `-+ permeability of the AMPA channel during de,:eloprnent. In vitro studies have documented Ca 2 permeable AMPA channels in neurons where GluR2 is underrepresented [57]. GluR2 mRNAs appear to rise more gradually during postnatal development and this is thought to play a role in the calcium permeability and relative hyperexcitability observed in neurons from immature animals [55]. The window of development which is characterized by enhanced excitatory neurotransmission coincides with a period during which inhibitory neurotransmission is relatively incomplete compared to the adult. GABA-mediated synaptic inhibition develops relatively late, increasing from birth until about 30 days in the rat [58-60]. It has long been known that GABA receptor binding also shows a similar increasing trend over the first weeks of life [61,62]. Furthermore, during the first postnatal week of life, GABA actually depolarizes immature hippocampal neurons by an outward flux of CI-, and towards the end of the first postnatal week its action shifts to a hyperpolarizing effect as seen in mature brain [63,64]. lntracellular ion homeostasis ",aries with age. Levels of calbindin D28K are lower in developing

137

hippocampaI ceBls than in adut~ tissue [65]~ which could result in increased concentrations of Ca -~' intracell~Jlarly under conditions which activate glutamate receptors [66]. In response ~o hypoxia~ intracellular K + rises more sJowty in immature brain than in adult, which may result in a window of opportunity for seizure activity before isoelectric state [67]. In addition, hippocampal neuronal N a / K pump activity is lower in the developing hippocampus compared to the adult [68]. The bioenergetic response to hypoxic stress is also dependent on maturation. Observations in the present in vivo rat model showed that global hypoxia resulted in a greater reduction in high energy phosphates in the immature rat (Pl0) than the adult, although this depletion was completely reversible upon reoxygenation [69]. This suggests that a given hypoxic insult depletes energy stores in the immature brain to a greater degree than in the adult. Given that the N a / K pump is dependent on ATP, depolarization may be prolonged in the immature brain in response to hypoxia, contribution to an epileptiform response. The cumulative effect of these maturational changes in receptor densities and subunit composition, as well as ion metabolism may increase intracellular calcium concentration. As increases in intracellular calcium mediate excitotoxicity and activation of second messenger systems and gene regulation [70-72], the immature brain may be more vulnerable than the adult to long term functional and structural consequences of seizure activity and hypoxia. Model systems such as those described here allow for correlation of in vitro and in vivo electrophysiology and should provide data regarding the pharmacological and physiological characteristics of hypoxia-induced seizure activity in the immature brain which could ultimately be applied to therapeutic strategies.

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