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Age dependence of NMDA receptor involvement in epileptiform activity in rat hippocampal slices
EPILEPSY RF EARCH ELSEVIER
Epilepsy Research 23 (1996) 105-113
Age dependence of NMDA receptor involvement in epileptiform activity in rat hippocampal slices Carl Wang, Frances E. Jensen
*
Department of Neurology, Children's Hospital, Boston, Massachusetts, USA
Received 20 July 1995; revised 26 September 1995; accepted 2 October 1995
Abstract The pattern of epileptiform activity recorded from a number of in vitro seizure models is age dependent: ictal discharges are observed in immature brain slices while interictal bursts are seen in adult brain slices. This study evaluated the involvement of the N-methyl-D-aspartate (NMDA) receptor in the age-dependency of epileptiform activity recorded in area CA1 of hippocampal slices in Mg2+-free medium. Incubation in Mg2+-free medium induced ictal activity in 84% of hippocampal slices from immature rats (postnatal 10-15 days). In contrast, adult slices responded with interictal bursting, while ictal activity was rare (9%). Bath application of the NMDA receptor antagonist D,L-2-amino-5-phosphonovaleric acid (DL-APV, 20 ~M) converted ictal activity to interictal activity in the hippocampal slices from immature rats. In adult slices, bath application of NMDA (10-20 /zM) in Mg2+-free medium induced ictal-like discharges. Perfusion with NMDA (20 /xM) in a medium containing 1.5 mM Mg 2÷ induced ictal activity in immature slices while it evoked only interictal bursts in adult slices. These results suggest that differences in NMDA receptor function may be involved in the age-dependency of epileptiform activity induced by Mg2+-free medium. Enhanced NMDA receptor-mediated activity may partially underlie increased seizure susceptibility in the immature brain. Keywords: Seizure susceptibility; N-methyl-D-aspartate(NMDA); Hippocampus; Development
1. Introduction Clinical evidence demonstrates that seizure incidence is increased early in life [1,21]. Consistent with this, increased seizure susceptibility is observed in immature animals under a variety of convulsant conditions, including electrical kindling [31,41], hy-
* Corresponding author. Department of Neurology, Children's Hospital, Enders 260, 300 Longwood Ave, Boston, MA 02115, USA. Tel.: (617) 355-8439; fax: (617) 738-1542.
poxia [26-28] and application of specific chemical convulsants such as kainic acid [3] pentylenetetrazol [47], picrotoxin [48], and N M D A [35]. Age dependent differences in the pattern of experimentally induced epileptiform activity have been shown in brain slices in vitro. In immature rat brain slices, electrographic seizures characterized by ictal discharges can be induced by perfusion with 4aminopyridine [6,11 ], tetraethylammonium [17], penicillin [43], picrotoxin [18,19], bicuculline [9] or low-Mg 2÷ medium [4,19,32,33]. In contrast, only interictal bursts are evoked by similar treatments in
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C. Wang, F.E. Jensen / Epilepsy Research 23 (1996) 105-113
adult slices [9,17,38,43-45]. In addition to age effects, the pattern of epileptiform activity is also region dependent. Heinemann and coworkers have demonstrated that low-Mg 2+ medium could induce ictal activity in the entorhinal cortex of adult rats, while only interictal activity was observed in the hippocampus [15,49]. The mechanisms underlying this age-dependent difference in the pattem of epileptiform activity remain to be elucidated. The N-methyl-D-aspartate (NMDA) receptor is important in the generation of epileptic activity [14]. Maturational differences in NMDA receptor-mediated activity may contribute to the hyperexcitability observed in the immature brain. The duration of NMDA receptor-mediated currents in the visual cortex [10] and superior colliculus [22] is longer during early development than in adulthood. The magnitude of the NMDA component of excitatory postsynaptic potentials (EPSPs) is higher in the visual cortex of 2-3 week-old rats than in older rats [29]. The amplitude of long-term potentiation in the hippocampus [20] and visual cortex [29] is greatest in 2-3 week-old rat pups, and this potentiation can be blocked by the NMDA antagonist 2amino-5-phosphonovaleric acid (APV) [25,29]. In addition, the neurotoxic effects of NMDA are enhanced between postnatal days (P) 4 and 14 in the rat [23,36]. Enhanced NMDA-mediated activity during early development may affect seizure susceptibility. The present study was undertaken to test the hypothesis that increased ictal activity observed in the immature brain is partially mediated by NMDA-mediated activity. First, epileptiform responses recorded in area CA1 in Mg2+-free medium were compared in hippocampal slices removed from P10-15 rats (the age window previously demonstrated as hyperexcitable) and adult rats. Second, the effect of the NMDA receptor antagonist APV on ictal activity recorded in area CA1 of immature slices was assessed, as well as the ability of NMDA to induce a pattern of ictal activity in adult slices in MgZ+-free medium. Third, responses in area CA1 to NMDA in a medium containing Mg 2+ were compared in immature and adult slices. Fourth, recordings were performed in CA1 and CA2-3 minislices from the immature brain to determine where the age-dependent ictal activity could be modulated.
2. Methods
Hippocampal slices were obtained from Long Evans rat pups (P10-15) and adult rats (200-400 g), and one to two slices from each animal were used. In preliminary experiments, we determined that the window of seizure susceptibility to Mg2+-free medium included the ages between P10 and P15. After decapitation, the brain was removed and submerged in cold (6-8°C) oxygenated artificial cerebrospinal fluid (ACSF, in raM: 124 NaCI, 5 KCI, 1.25 NaH2PO 4, 24 NaHCO 3, 1.5 MgCI 2, 1.8 CaCI: and 10 D-glucose, pH 7.4). On an ice-cooled plate, the hemispheres were separated by a sagittal cut through the middle of the corpus callosum. The left hippocampus was removed following two transverse cuts across the end of septal and temporal poles. Transverse hippocampal slices, 400 /zm thick, were cut from the temporal pole using a tissue chopper (Stoelting). In some experiments, CA2-3 and CA1 minislices were made by an incision between area CA2-3 and area CA1/dentate gyms. The slices were then transferred to an interface chamber and maintained at the interface between ACSF and 95% 0 2 - 5 % CO 2 for at least 1 h before recording began. To induce epileptiform activity, normal ACSF (containing 1.5 mM Mg 2+) was switched to Mg2+-free ACSF. Adult slices were perfused with ACSF (40 m l / h ) at 37°C while pup slices were perfused at 33°C to reflect the age differences in body temperature in vivo. As an additional control for temperature effects, another group of immature slices were evaluated at 37°C. Extracellular field potentials were recorded mainly at the CA1 pyramidal cell body layer (stratum pyramidale), except in the minislice study recordings were performed in either area CA1 or CA3. Glass microelectrodes filled with 1 M NaC! (resistance 2 - 4 M ~ ) were used for recording. Signals were amplified in AC mode by an AC amplifier (A-M Systems), monitored on an oscilloscope (Tektronix), and recorded by a chart recorder (Gould). DL-APV (Sigma, 2.5-100 p,M) and NMDA (Sigma, 5-20 /zM) were dissolved in the perfusion medium and administered by bath application. APV and NMDA were each applied for 20-30 rain. Effects of drugs on the incidence of electrographic seizures were analyzed by chi-square test or
C. Wang, F.E. Jensen /Epilepsy Research 23 (1996) 105-113
Fisher's Exact test. The effects of drugs on the duration and interval of seizures were analyzed by Student's t-test.
3. Results
Within 30 min of l:erfusion with Mg 2+-free ACSF, spontaneous ictal discharges were observed in 27/32 immature slices (one slice from each pup). Several interictal bursts were usually seen preceding ictal events (Fig. 1). During a 15-45-min recording period, the duration of individual episodes of ictal activity ranged from 6 to 68 s and the interval between ictal episodes ranged from 1.6 to 4.5 min. In contrast, adult slices incubated in Mg2+-free ACSF typically exhibited continuous interictal bursts (Fig. 1), and ictal discharges were seen in only 3 / 3 4 slices (1-2 slices from each rat), which was significantly less frequent compared to immature slices (P < 0.0001, chi-square test). Another group of im-
107
mature slices was perfused at 37°C and MgZ+-free ACSF induced ictal activity in 20/25 slices, which was similar to the result at 33°C, and significantly more frequent than in adult slices (P < 0.0001, chisquare test). DL-APV was administered after ictal activity was induced in immature slices in Mg2+-free ACSF for at least 15 min. A concentration of 2.5/xM DL-APV blocked ictal activity in 4 / 8 immature slices, and in the slices which still had ictal activity there was a significant increase in the interval between ictal events (from 3.08 + 0.27 min to 4.92 _+ 0.67 min, P < 0.05, paired Student's t-test). Ictal activity was completely blocked in l 1/12 immature slices by bath application of 20 /xM DL-APV, and in these slices rhythmic interictal spikes emerged after the disappearance of ictal discharges (Fig. 2). Spontaneous interictal bursts persisted in immature slices (n = 6) in Mg2+-free medium at an even higher concentration of DL-APV (100/zM) during a 30-min observation period.
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Fig. 1. Mg2+-free ACSF induced age-dependent epileptiform activity in area CA1 of rat hippocampal slices. Top panel: ictal activity recorded in an immature slice from a l0 day-old rat. Bottom panel: interictal activity recorded in an adult slice.
108
C. Wang, F.E, Jensen / Epilepsy Research 23 (1996) 105-113 Control
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Fig. 2. Effect of DL-APV on the pattern of epileptiform activity in area CA1 of an immature hippocampal slice from a 12 day-old rat in Mg2+-free ACSF. Top trace: spontaneous ictal discharges preceded by a few interictal bursts before APV application. Middle trace: five minutes after perfusion with 20 /~M DL-APV, the ictal pattern was converted to an interictal pattern. Bottom trace: five minutes after washout of APV, the ictal pattern reappeared.
In adult slices exhibiting interictal bursts in Mg2÷-free ACSF, addition of 10 or 20 /xM NMDA induced ictal-like discharges in 4 / 8 and 9 / 1 2 adult slices, respectively. In two slices perfused with 10 ~zM NMDA, recurrent ictal-like events were observed (Fig. 3). Depression occurred following NMDA-induced seizures in most slices which experienced ictal activity, except only one in the 10-/xM
NMDA group, and continued throughout NMDA application. Application of NMDA in ACSF containing 1.5 mM Mg 2÷ induced age-dependent epileptiform responses. In immature slices, 5 /zM NMDA induced interictal activity in only 1/10 slices. At 10 /xM NMDA, interictal bursting was seen in 8 / 1 0 slices. When the concentration of NMDA was increased to
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Fig. 3. Ictal-like discharges induced by perfusion of NMDA in area CAI of an adult hippocampal slice in Mg2+-free ACSF. Top trace: interictal bursts before NMDA application. Middle trace: ictal-like discharges occurred 10 min after perfusion with 10 /LM NMDA. Bottom trace: ten minutes after washout of NMDA, interictal bursts recurred.
C. Wang, F.E. Jensen / Epilepsy Research 23 (1996) 105-113
20 ~M, ictal activity was seen in all slices tested (Fig. 4, Table 1). In contrast, the threshold for NMDA-induced interictal bursting was higher in adult slices, with 10 /zM producing no epileptiform activity and interictal bursting seen in 7/12 slices at 20 ~ M NMDA (Fig. 4, Table 1). Hippocampal minislices were used to determine where the pattern of epileptiform activity seen in immature slices could be modulated by APV. Since the epileptiform activity induced by zero Mg 2+ may
Table 1 Epileptiform activity induced by NMDA in ACSF containing 1.5 mM Mg 2+ in immature and adult hippocampal slices NMDA (/xM)
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Fig. 4. Responses in area CA1 of immature and adult slices to bath application of NMDA in ACSF containing 1.5 mM Mg 2+. Top panel: responses of immature slices from three 13-14 day-old rats to NMDA (5-20 p.M). Bottom panel: responses of adult slices to NMDA (10-20 /.~M).
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C. Wang, F.E. Jensen / Epilepsy Research 23 (1996) 105-113
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1 rain Fig. 5. Effect of DL-APV on the pattern of epileptiform activity in area CA3 of an immature CA2-3 hippocampal minislice from a 12 day-old rat bathed in Mg2+-free ACSF. Top trace: spontaneous ictal discharges preceded by several interictal bursts before APV application. Middle trace: ictal activity was changed to interictal activity 5 min after perfusion with 20 /zM DL-APV. Bottom trace: ten minutes after washout of APV, ictal activity reappeared.
persist after switching to normal ACSF [32], only naive minislices from immature rats were used in this study. First, the responses of area CA1 and CA3 to Mg2+-free ACSF were compared in CA1 and CA2-3 minislices cut from the same slice (n = 10 pairs). CA2-3 minislices were found to be more susceptible to Mg2+-free ACSF, as epileptiform activity was recorded in area CA3 of all CA2-3 minislices (7 ictal/3 interictal) while epileptiform activity was observed in area CA1 of 5 / 1 0 CA1 minislices (2 ictal/3 interictal). A subsequent experiment was carried out in CA2-3 minislices displaying ictal activity (n = 11). Bath application of 20 /xM DLAPV blocked ictal activity in area CA3 and induced continuous rhythmic interictal bursts in 10/11 minislices (Fig. 5). 4. Discussion
This study demonstrates that (1) the ictal activity induced by Mg2+-free ACSF in immature hippocampal slices can be converted to interictal activity by the NMDA receptor antagonist APV, (2) application of NMDA in Mg2+-free ACSF could induce ictal-like discharges in adult hippocampal slices, (3) immature slices are more susceptible to NMDA-induced ictal activity than adult slices and (4) pattern transition
from ictal to interictal by APV application could occur in area CA3. It has been previously shown that perfusion with low-Mg 2+ ACSF induces interictal bursts in adult hippocampal slices [38,44,45], while identical conditions induce ictal discharges in immature hippocampal slices [4,19,32,33]. Interictal and ictal activity do not develop when D-APV is present prior to and during exposure to zero-Mg 2+ ACSF [32], indicating that the NMDA receptor is involved in generating both interictal and ictal activity. The present study suggests that the difference in NMDA receptormediated transmission in adult and immature brains may in part be responsible for the age-dependent pattern of epileptiform activity. In immature slices exhibiting ictal activity, the NMDA receptor antagonist APV was able to convert the ictal (immature) pattern to the interictal (adul0 pattern. Preliminary results demonstrate that other methods of modulating NMDA receptor-mediated activity, such as agents acting at the NMDA receptor redox sites [2], are capable of switching low-Mg 2+ induced ictal activity to interictal activity in immature slices [50]. In the present study, overactivation of NMDA receptors by application of NMDA in Mg2+-free ACSF could induce ictal-like activity in adult slices. The results suggest that age-dependent differences in the pattern
C. Wang, F.E. Jensen / Epilepsy Research 23 (1996) 105-113
of epileptiform activity induced by Mg2+-free ACSF may be caused by an enhanced NMDA-mediated activity. If immature slices have a higher NMDA mediated activity than adult slices, then the application of NMDA may induce a greater response in immature slices than in adult slices. The present study demonstrates that the threshold for NMDA-induced ictal activity was lower in immature slices than in adult slices. NMDA at 20 /xM induced ictal activity in immature but not in adult slices. In this study higher NMDA concentrations were not used, but others report that application of NMDA at concentrations of 5 0 - 1 0 0 / z M abolishes the EPSP [5]. Taken together, these results suggest that immature slices may have enhanced NMDA receptor-mediated transmission, which may contribul:e to the enhanced seizure susceptibility of the imraature brain. In the present st~Ldy, DL-APV (up to 100 /xM) failed to block spontaneous bursting completely. A D-APV-resistant depolarizing component induced by Mg2+-free solution has been found to be blocked by the non-NMDA receptor antagonist 6-cyano-7nitroquinoxaline-2,3-dione [16]. These results suggest that non-NMDA receptors may be involved in the interictal bursting. Several maturational factors might contribute to enhanced NMDA-mediated activity in the immature brain. First, a substantial increase in the density of NMDA receptors has been found in the rat hippocampus [37,46] and cortex [24] during early development, and this may cause an increase in the NMDA-mediated postsynaptic response. Second, blockade of the NMDA receptor channel by Mg 2÷ is reduced in the immature rat hippocampus [8,40] and visual cortex [30], which may result in decreased voltage dependence of NMDA currents in immature neurons and lead to increased Ca 2+ influx [7]. The channel assembled fi'om NR1 and NR2C NMDA receptor subunits has been shown to be less sensitive to the blocking action of Mg 2+ [39]. The mRNA of NR2C is not observed in the adult hippocampus, but clearly detected in the 7 and 14 day-old rat hippocampal formation [42] and thus the low sensitivity to Mg 2+ may be ~!ue to expression of different NMDA receptor subunits during development. These maturational changes in subunit composition of NMDA receptors may be a factor in the increased
111
seizure susceptibility in the immature brain as evidenced by ictal activity. The pattern of epileptiform activity recorded in brain slices is not just age dependent but also region dependent. Studies using simultaneous multiple recording electrodes have demonstrated that lowMg 2÷ medium induces ictal activity in the entorhinal cortex, but interictal activity in area CA1 and CA3 of combined cortical-hippocampal slices from adult rats [15]. In the present study, low-Mg2+-induced epileptiform activity was seen more frequently in area CA3 than in area CA1 of immature slices. These results were consistent with previous studies showing that interruption of the connection between area CA3 and CA1 in adult slices blocks low-Mg2+-induced interictal activity in CA1, but epileptiform activity in area CA3 persists [38,44]. Local excitatory connections have been shown to exist between hippocampal pyramidal neurons and the connections are more dense in CA3 than in CA1 [12,13,34]. This study demonstrates that ictal activity in area CA3 of immature slices could be converted to interictal activity by APV. Taken together, the findings indicate that area CA3 may be a site generating epileptiform activity in both immature and adult slices and that NMDAmediated synaptic transmission in local circuits within CA3 may play an important role in regulating epileptiform activity. The results of this study provide in vitro evidence that enhanced NMDA-mediated function may underlie age dependent differences in hippocampal seizure susceptibility. Specific mechanisms for this enhancement are not yet known. However, these results are consistent with other studies that implicate an agedependent increase in other NMDA-mediated phenomena in the immature brain, such as LTP, excitotoxicity, and plasticity. It is possible that these agedependent phenomena may have similar underlying mechanisms to that governing the differences in hippocampal seizure susceptibility observed in immature and adult brains.
Acknowledgements This work was supported by NIH grants NS31718 and NS07264, and in part by a Mental Retardation Center Grant (P30-HD18655).
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[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
epileptiform activity induced by magnesium-free medium: Differences between areas CA I and CA2-3, Epilepsy Res., 6 (1990) 95-101. MacVicar, B.A. and Dudek, F.E., Local synaptic circuits in rat hippocampus: Interactions between pyramidal cells, Brain Res., 184 (1980) 220--223. Mare~, P. and Veli~ek, L., N-methyl-D-aspartate (NMDA)induced seizures in developing rats, Dev. Brain Res., 65 (1992) 185-189. McDonald, J.W., Silverstein, F.S. and Johnston, M.V., Neurotoxicity of N-methyI-D-aspartate is markedly enhanced in developing rat central aervous system, Brain Res., 459 (I 988) 200-203. McDonald, J.W., Johnston, M.V. and Young, A.B., Differential ontogenic development of three receptors comprising the NMDA receptor/channel complex in the rat hippocampus, Exp. Neurol., 110 (1990) 237-247. Mody, 1., Lambert, J.D.C. and Heinemann, U., Low extracellular magnesium induces epileptiform activity and spreading depression in rat hippocampal slices, J. Neurophysiol., 57 (1987) 869-888. Monyer, H., Sprengel, R., Schoepfer, R., Herb, A., Higuchi, M., Lomeli, H., Burnashev, N., Sakmann, B. and Seeburg, P.H., Heteromeric NMDA receptors: Molecular and functional distinction of .,;ubtypes, Science, 256 (1992) 12171221. Morrisett, R.A., Mott, D.D., Lewis, D.V., Wilson, W.A. and Swartzwelder, H.S., Reduced sensitivity of the N-methyl-oaspartate component ~zf synaptic transmission to magnesium in hippocampal slices from immature rats, Dev. Brain Res., 56 (1990) 257-262. Mosh6, S.L., Albala, E;.J., Ackermann, R.F. and Engel, J. Jr., Increased seizure susceptibility of the immature brain, Dev. Brain Res., 7 (1983) 81-85.
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[42] Pollard, H., Khrestchatisky, M., Moreau, J. and Ben-Ari, Y., Transient expression of the NR2C subunit of the NMDA receptor in developing rat brain, Neuroreport, 4 (1993) 411-414. [43] Swann, J.W. and Brady, R.J., Penicillin-induced epileptogenesis in immature rat CA3 hippocampal pyramidal cells, Dev. Brain Res., 12 (1984) 243-254. [44] Tancredi, V., Avoli, M. and Hwa, G.G.C., Low-magnesium epilepsy in rat hippocampal slices: Inhibitory postsynaptic potentials in the CAI subfield, Neurosci. Len., 89 (1988) 293-298. [45] Tancredi, V., Hwa, G.G.C., Zona, C., Brancati, A. and Avoli, M., Low magnesium epileptogenesis in the rat hippocampal slice: Electrophysiological and pharmacological features, Brain Res., 511 (1990) 280-290. [46] Tremblay, E., Roisin, M.P., Represa, A., CharriautMarlangue, C. and Ben-Ari, Y., Transient increased density of NMDA binding sites in the developing rat hippocampus, Brain Res., 46l (1988) 393-396. [47] Velisek, L., Kubova, H., Pohl, M., Stankova, L., Mare~;, P. and Schickerova, R., Pentylenetetrazol-induced seizures in rats: An ontogenetic study, Naunyn-Schmiedeberg's Arch. Pharmacol., 346 (1992) 588-591. [48] Vemadakis, A. and Woodbury, D.M., The developing animal as a model, Epilepsia, 10 (1969) 163-178. [49] Walther, H., Lambert, J.D.C., Jones, R.S.G., Heinemann, U. and Hamon, B., Epileptiform activity in combined slices of the hippocampus, subiculum and entorhinal cortex during perfusion with low magnesium medium, Neurosci. Lett., 69 (1986) 156-161. [50] Wang, C. and Jensen, F.E., Effects of redox manipulations on low magnesium-induced epileptiform activity in immature rat hippocampal slices, Soc. Neurosci. Abstr., 21 (1995) 984.
Epilepsy Research 23 (1996) 105-113
Age dependence of NMDA receptor involvement in epileptiform activity in rat hippocampal slices Carl Wang, Frances E. Jensen
*
Department of Neurology, Children's Hospital, Boston, Massachusetts, USA
Received 20 July 1995; revised 26 September 1995; accepted 2 October 1995
Abstract The pattern of epileptiform activity recorded from a number of in vitro seizure models is age dependent: ictal discharges are observed in immature brain slices while interictal bursts are seen in adult brain slices. This study evaluated the involvement of the N-methyl-D-aspartate (NMDA) receptor in the age-dependency of epileptiform activity recorded in area CA1 of hippocampal slices in Mg2+-free medium. Incubation in Mg2+-free medium induced ictal activity in 84% of hippocampal slices from immature rats (postnatal 10-15 days). In contrast, adult slices responded with interictal bursting, while ictal activity was rare (9%). Bath application of the NMDA receptor antagonist D,L-2-amino-5-phosphonovaleric acid (DL-APV, 20 ~M) converted ictal activity to interictal activity in the hippocampal slices from immature rats. In adult slices, bath application of NMDA (10-20 /zM) in Mg2+-free medium induced ictal-like discharges. Perfusion with NMDA (20 /xM) in a medium containing 1.5 mM Mg 2÷ induced ictal activity in immature slices while it evoked only interictal bursts in adult slices. These results suggest that differences in NMDA receptor function may be involved in the age-dependency of epileptiform activity induced by Mg2+-free medium. Enhanced NMDA receptor-mediated activity may partially underlie increased seizure susceptibility in the immature brain. Keywords: Seizure susceptibility; N-methyl-D-aspartate(NMDA); Hippocampus; Development
1. Introduction Clinical evidence demonstrates that seizure incidence is increased early in life [1,21]. Consistent with this, increased seizure susceptibility is observed in immature animals under a variety of convulsant conditions, including electrical kindling [31,41], hy-
* Corresponding author. Department of Neurology, Children's Hospital, Enders 260, 300 Longwood Ave, Boston, MA 02115, USA. Tel.: (617) 355-8439; fax: (617) 738-1542.
poxia [26-28] and application of specific chemical convulsants such as kainic acid [3] pentylenetetrazol [47], picrotoxin [48], and N M D A [35]. Age dependent differences in the pattern of experimentally induced epileptiform activity have been shown in brain slices in vitro. In immature rat brain slices, electrographic seizures characterized by ictal discharges can be induced by perfusion with 4aminopyridine [6,11 ], tetraethylammonium [17], penicillin [43], picrotoxin [18,19], bicuculline [9] or low-Mg 2÷ medium [4,19,32,33]. In contrast, only interictal bursts are evoked by similar treatments in
0920-1211/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0920-1 211(95)00086-0
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C. Wang, F.E. Jensen / Epilepsy Research 23 (1996) 105-113
adult slices [9,17,38,43-45]. In addition to age effects, the pattern of epileptiform activity is also region dependent. Heinemann and coworkers have demonstrated that low-Mg 2+ medium could induce ictal activity in the entorhinal cortex of adult rats, while only interictal activity was observed in the hippocampus [15,49]. The mechanisms underlying this age-dependent difference in the pattem of epileptiform activity remain to be elucidated. The N-methyl-D-aspartate (NMDA) receptor is important in the generation of epileptic activity [14]. Maturational differences in NMDA receptor-mediated activity may contribute to the hyperexcitability observed in the immature brain. The duration of NMDA receptor-mediated currents in the visual cortex [10] and superior colliculus [22] is longer during early development than in adulthood. The magnitude of the NMDA component of excitatory postsynaptic potentials (EPSPs) is higher in the visual cortex of 2-3 week-old rats than in older rats [29]. The amplitude of long-term potentiation in the hippocampus [20] and visual cortex [29] is greatest in 2-3 week-old rat pups, and this potentiation can be blocked by the NMDA antagonist 2amino-5-phosphonovaleric acid (APV) [25,29]. In addition, the neurotoxic effects of NMDA are enhanced between postnatal days (P) 4 and 14 in the rat [23,36]. Enhanced NMDA-mediated activity during early development may affect seizure susceptibility. The present study was undertaken to test the hypothesis that increased ictal activity observed in the immature brain is partially mediated by NMDA-mediated activity. First, epileptiform responses recorded in area CA1 in Mg2+-free medium were compared in hippocampal slices removed from P10-15 rats (the age window previously demonstrated as hyperexcitable) and adult rats. Second, the effect of the NMDA receptor antagonist APV on ictal activity recorded in area CA1 of immature slices was assessed, as well as the ability of NMDA to induce a pattern of ictal activity in adult slices in MgZ+-free medium. Third, responses in area CA1 to NMDA in a medium containing Mg 2+ were compared in immature and adult slices. Fourth, recordings were performed in CA1 and CA2-3 minislices from the immature brain to determine where the age-dependent ictal activity could be modulated.
2. Methods
Hippocampal slices were obtained from Long Evans rat pups (P10-15) and adult rats (200-400 g), and one to two slices from each animal were used. In preliminary experiments, we determined that the window of seizure susceptibility to Mg2+-free medium included the ages between P10 and P15. After decapitation, the brain was removed and submerged in cold (6-8°C) oxygenated artificial cerebrospinal fluid (ACSF, in raM: 124 NaCI, 5 KCI, 1.25 NaH2PO 4, 24 NaHCO 3, 1.5 MgCI 2, 1.8 CaCI: and 10 D-glucose, pH 7.4). On an ice-cooled plate, the hemispheres were separated by a sagittal cut through the middle of the corpus callosum. The left hippocampus was removed following two transverse cuts across the end of septal and temporal poles. Transverse hippocampal slices, 400 /zm thick, were cut from the temporal pole using a tissue chopper (Stoelting). In some experiments, CA2-3 and CA1 minislices were made by an incision between area CA2-3 and area CA1/dentate gyms. The slices were then transferred to an interface chamber and maintained at the interface between ACSF and 95% 0 2 - 5 % CO 2 for at least 1 h before recording began. To induce epileptiform activity, normal ACSF (containing 1.5 mM Mg 2+) was switched to Mg2+-free ACSF. Adult slices were perfused with ACSF (40 m l / h ) at 37°C while pup slices were perfused at 33°C to reflect the age differences in body temperature in vivo. As an additional control for temperature effects, another group of immature slices were evaluated at 37°C. Extracellular field potentials were recorded mainly at the CA1 pyramidal cell body layer (stratum pyramidale), except in the minislice study recordings were performed in either area CA1 or CA3. Glass microelectrodes filled with 1 M NaC! (resistance 2 - 4 M ~ ) were used for recording. Signals were amplified in AC mode by an AC amplifier (A-M Systems), monitored on an oscilloscope (Tektronix), and recorded by a chart recorder (Gould). DL-APV (Sigma, 2.5-100 p,M) and NMDA (Sigma, 5-20 /zM) were dissolved in the perfusion medium and administered by bath application. APV and NMDA were each applied for 20-30 rain. Effects of drugs on the incidence of electrographic seizures were analyzed by chi-square test or
C. Wang, F.E. Jensen /Epilepsy Research 23 (1996) 105-113
Fisher's Exact test. The effects of drugs on the duration and interval of seizures were analyzed by Student's t-test.
3. Results
Within 30 min of l:erfusion with Mg 2+-free ACSF, spontaneous ictal discharges were observed in 27/32 immature slices (one slice from each pup). Several interictal bursts were usually seen preceding ictal events (Fig. 1). During a 15-45-min recording period, the duration of individual episodes of ictal activity ranged from 6 to 68 s and the interval between ictal episodes ranged from 1.6 to 4.5 min. In contrast, adult slices incubated in Mg2+-free ACSF typically exhibited continuous interictal bursts (Fig. 1), and ictal discharges were seen in only 3 / 3 4 slices (1-2 slices from each rat), which was significantly less frequent compared to immature slices (P < 0.0001, chi-square test). Another group of im-
107
mature slices was perfused at 37°C and MgZ+-free ACSF induced ictal activity in 20/25 slices, which was similar to the result at 33°C, and significantly more frequent than in adult slices (P < 0.0001, chisquare test). DL-APV was administered after ictal activity was induced in immature slices in Mg2+-free ACSF for at least 15 min. A concentration of 2.5/xM DL-APV blocked ictal activity in 4 / 8 immature slices, and in the slices which still had ictal activity there was a significant increase in the interval between ictal events (from 3.08 + 0.27 min to 4.92 _+ 0.67 min, P < 0.05, paired Student's t-test). Ictal activity was completely blocked in l 1/12 immature slices by bath application of 20 /xM DL-APV, and in these slices rhythmic interictal spikes emerged after the disappearance of ictal discharges (Fig. 2). Spontaneous interictal bursts persisted in immature slices (n = 6) in Mg2+-free medium at an even higher concentration of DL-APV (100/zM) during a 30-min observation period.
Immature
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Fig. 1. Mg2+-free ACSF induced age-dependent epileptiform activity in area CA1 of rat hippocampal slices. Top panel: ictal activity recorded in an immature slice from a l0 day-old rat. Bottom panel: interictal activity recorded in an adult slice.
108
C. Wang, F.E, Jensen / Epilepsy Research 23 (1996) 105-113 Control
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Fig. 2. Effect of DL-APV on the pattern of epileptiform activity in area CA1 of an immature hippocampal slice from a 12 day-old rat in Mg2+-free ACSF. Top trace: spontaneous ictal discharges preceded by a few interictal bursts before APV application. Middle trace: five minutes after perfusion with 20 /~M DL-APV, the ictal pattern was converted to an interictal pattern. Bottom trace: five minutes after washout of APV, the ictal pattern reappeared.
In adult slices exhibiting interictal bursts in Mg2÷-free ACSF, addition of 10 or 20 /xM NMDA induced ictal-like discharges in 4 / 8 and 9 / 1 2 adult slices, respectively. In two slices perfused with 10 ~zM NMDA, recurrent ictal-like events were observed (Fig. 3). Depression occurred following NMDA-induced seizures in most slices which experienced ictal activity, except only one in the 10-/xM
NMDA group, and continued throughout NMDA application. Application of NMDA in ACSF containing 1.5 mM Mg 2÷ induced age-dependent epileptiform responses. In immature slices, 5 /zM NMDA induced interictal activity in only 1/10 slices. At 10 /xM NMDA, interictal bursting was seen in 8 / 1 0 slices. When the concentration of NMDA was increased to
co~t~o~_ ~ M ~ H ~ i ~ i ~ h ~ j ~ iI~ L ~ i ~ t11~t~ ~"I~ii~i~t~L~i~i~i~i~i~t~ii~
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Fig. 3. Ictal-like discharges induced by perfusion of NMDA in area CAI of an adult hippocampal slice in Mg2+-free ACSF. Top trace: interictal bursts before NMDA application. Middle trace: ictal-like discharges occurred 10 min after perfusion with 10 /LM NMDA. Bottom trace: ten minutes after washout of NMDA, interictal bursts recurred.
C. Wang, F.E. Jensen / Epilepsy Research 23 (1996) 105-113
20 ~M, ictal activity was seen in all slices tested (Fig. 4, Table 1). In contrast, the threshold for NMDA-induced interictal bursting was higher in adult slices, with 10 /zM producing no epileptiform activity and interictal bursting seen in 7/12 slices at 20 ~ M NMDA (Fig. 4, Table 1). Hippocampal minislices were used to determine where the pattern of epileptiform activity seen in immature slices could be modulated by APV. Since the epileptiform activity induced by zero Mg 2+ may
Table 1 Epileptiform activity induced by NMDA in ACSF containing 1.5 mM Mg 2+ in immature and adult hippocampal slices NMDA (/xM)
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* P < 0.05 compared to the adult slice group bathed in the same concentration of NMDA (Fisher's Exact test).
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Fig. 4. Responses in area CA1 of immature and adult slices to bath application of NMDA in ACSF containing 1.5 mM Mg 2+. Top panel: responses of immature slices from three 13-14 day-old rats to NMDA (5-20 p.M). Bottom panel: responses of adult slices to NMDA (10-20 /.~M).
110
C. Wang, F.E. Jensen / Epilepsy Research 23 (1996) 105-113
Control
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1 rain Fig. 5. Effect of DL-APV on the pattern of epileptiform activity in area CA3 of an immature CA2-3 hippocampal minislice from a 12 day-old rat bathed in Mg2+-free ACSF. Top trace: spontaneous ictal discharges preceded by several interictal bursts before APV application. Middle trace: ictal activity was changed to interictal activity 5 min after perfusion with 20 /zM DL-APV. Bottom trace: ten minutes after washout of APV, ictal activity reappeared.
persist after switching to normal ACSF [32], only naive minislices from immature rats were used in this study. First, the responses of area CA1 and CA3 to Mg2+-free ACSF were compared in CA1 and CA2-3 minislices cut from the same slice (n = 10 pairs). CA2-3 minislices were found to be more susceptible to Mg2+-free ACSF, as epileptiform activity was recorded in area CA3 of all CA2-3 minislices (7 ictal/3 interictal) while epileptiform activity was observed in area CA1 of 5 / 1 0 CA1 minislices (2 ictal/3 interictal). A subsequent experiment was carried out in CA2-3 minislices displaying ictal activity (n = 11). Bath application of 20 /xM DLAPV blocked ictal activity in area CA3 and induced continuous rhythmic interictal bursts in 10/11 minislices (Fig. 5). 4. Discussion
This study demonstrates that (1) the ictal activity induced by Mg2+-free ACSF in immature hippocampal slices can be converted to interictal activity by the NMDA receptor antagonist APV, (2) application of NMDA in Mg2+-free ACSF could induce ictal-like discharges in adult hippocampal slices, (3) immature slices are more susceptible to NMDA-induced ictal activity than adult slices and (4) pattern transition
from ictal to interictal by APV application could occur in area CA3. It has been previously shown that perfusion with low-Mg 2+ ACSF induces interictal bursts in adult hippocampal slices [38,44,45], while identical conditions induce ictal discharges in immature hippocampal slices [4,19,32,33]. Interictal and ictal activity do not develop when D-APV is present prior to and during exposure to zero-Mg 2+ ACSF [32], indicating that the NMDA receptor is involved in generating both interictal and ictal activity. The present study suggests that the difference in NMDA receptormediated transmission in adult and immature brains may in part be responsible for the age-dependent pattern of epileptiform activity. In immature slices exhibiting ictal activity, the NMDA receptor antagonist APV was able to convert the ictal (immature) pattern to the interictal (adul0 pattern. Preliminary results demonstrate that other methods of modulating NMDA receptor-mediated activity, such as agents acting at the NMDA receptor redox sites [2], are capable of switching low-Mg 2+ induced ictal activity to interictal activity in immature slices [50]. In the present study, overactivation of NMDA receptors by application of NMDA in Mg2+-free ACSF could induce ictal-like activity in adult slices. The results suggest that age-dependent differences in the pattern
C. Wang, F.E. Jensen / Epilepsy Research 23 (1996) 105-113
of epileptiform activity induced by Mg2+-free ACSF may be caused by an enhanced NMDA-mediated activity. If immature slices have a higher NMDA mediated activity than adult slices, then the application of NMDA may induce a greater response in immature slices than in adult slices. The present study demonstrates that the threshold for NMDA-induced ictal activity was lower in immature slices than in adult slices. NMDA at 20 /xM induced ictal activity in immature but not in adult slices. In this study higher NMDA concentrations were not used, but others report that application of NMDA at concentrations of 5 0 - 1 0 0 / z M abolishes the EPSP [5]. Taken together, these results suggest that immature slices may have enhanced NMDA receptor-mediated transmission, which may contribul:e to the enhanced seizure susceptibility of the imraature brain. In the present st~Ldy, DL-APV (up to 100 /xM) failed to block spontaneous bursting completely. A D-APV-resistant depolarizing component induced by Mg2+-free solution has been found to be blocked by the non-NMDA receptor antagonist 6-cyano-7nitroquinoxaline-2,3-dione [16]. These results suggest that non-NMDA receptors may be involved in the interictal bursting. Several maturational factors might contribute to enhanced NMDA-mediated activity in the immature brain. First, a substantial increase in the density of NMDA receptors has been found in the rat hippocampus [37,46] and cortex [24] during early development, and this may cause an increase in the NMDA-mediated postsynaptic response. Second, blockade of the NMDA receptor channel by Mg 2÷ is reduced in the immature rat hippocampus [8,40] and visual cortex [30], which may result in decreased voltage dependence of NMDA currents in immature neurons and lead to increased Ca 2+ influx [7]. The channel assembled fi'om NR1 and NR2C NMDA receptor subunits has been shown to be less sensitive to the blocking action of Mg 2+ [39]. The mRNA of NR2C is not observed in the adult hippocampus, but clearly detected in the 7 and 14 day-old rat hippocampal formation [42] and thus the low sensitivity to Mg 2+ may be ~!ue to expression of different NMDA receptor subunits during development. These maturational changes in subunit composition of NMDA receptors may be a factor in the increased
111
seizure susceptibility in the immature brain as evidenced by ictal activity. The pattern of epileptiform activity recorded in brain slices is not just age dependent but also region dependent. Studies using simultaneous multiple recording electrodes have demonstrated that lowMg 2÷ medium induces ictal activity in the entorhinal cortex, but interictal activity in area CA1 and CA3 of combined cortical-hippocampal slices from adult rats [15]. In the present study, low-Mg2+-induced epileptiform activity was seen more frequently in area CA3 than in area CA1 of immature slices. These results were consistent with previous studies showing that interruption of the connection between area CA3 and CA1 in adult slices blocks low-Mg2+-induced interictal activity in CA1, but epileptiform activity in area CA3 persists [38,44]. Local excitatory connections have been shown to exist between hippocampal pyramidal neurons and the connections are more dense in CA3 than in CA1 [12,13,34]. This study demonstrates that ictal activity in area CA3 of immature slices could be converted to interictal activity by APV. Taken together, the findings indicate that area CA3 may be a site generating epileptiform activity in both immature and adult slices and that NMDAmediated synaptic transmission in local circuits within CA3 may play an important role in regulating epileptiform activity. The results of this study provide in vitro evidence that enhanced NMDA-mediated function may underlie age dependent differences in hippocampal seizure susceptibility. Specific mechanisms for this enhancement are not yet known. However, these results are consistent with other studies that implicate an agedependent increase in other NMDA-mediated phenomena in the immature brain, such as LTP, excitotoxicity, and plasticity. It is possible that these agedependent phenomena may have similar underlying mechanisms to that governing the differences in hippocampal seizure susceptibility observed in immature and adult brains.
Acknowledgements This work was supported by NIH grants NS31718 and NS07264, and in part by a Mental Retardation Center Grant (P30-HD18655).
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