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Extracellular K+ and Ca2+ changes during epileptiform discharges in the immature rat neocortex
Developmental Brain Research, 36 (1987) 299-303
299
Elsevier BRD 60239
Extracellular K + and Ca 2+ changes during epileptiform discharges in the immature rat neocortex John J. Hablitz and Uwe Heinemann* Section of Neurophysiology, Departmentof Neurology, Baylor Collegeof Medicine, Houston, Texas 77030(U.S.A.) and Department of Neurophysiology, Max-PlanckInstitutefor Psychiatry, Planegg-Martinsried(F.R.G.) (Accepted 18 August 1987)
Key words: Neocortex; Epileptiform activity; Extracellular potassium; Extracellular calcium; Development
Picrotoxin-induced epileptiform activity was examined in neocortical slices prepared from 8- to 15-day-old rats. This activity consisted of spontaneous bursts of 3-5 discharges that resembled interictal spikes and were interspersed with ictal-like paroxysms lasting 10-30 s. Measurements of extracellular potassium ([K+]o) and calcium ([Ca2+]o)were made during these spontaneous epileptiform events, using ion-sensitive electrodes. Individual interictal spikes were associated with [Ca2+]odecreases of 0,1-0.2 mM, whereas sustained ictal-like discharges were accompanied by decreases of 0.3-0.4 mM. Measurement of [K+]o showed that individual interictal spikes were associated with increases in [K+]oup to t2 raM, whereas increases to more than 20 mM accompanied long-lasting ictal-like discharges. Maximum increases in [K+]o were observed ca. 600 pm below the pial surface. [K+]o increases were followed by undershoots of the resting [K÷]o level. The unusually high [K÷]o levels associated with epileptiform discharges in the immature neocortex suggest that disturbances in [K÷]o regulation may contribute to the generation of the picrotoxin-induced, spontaneous, prolonged ictal-like discharges observed in the 8- to 15-day age group.
The incidence of seizure disorders in early life is quite high in man, and several unique forms of epilepsy occur in the neonatal period iS. Experimental studies in neonatal rats have demonstrated that the brain is more prone to manifest epileptiform discharges at certain times during development j2' Is~'). Studies in vitro have documented that during postnatal development, there is a stage in which certain regions of the brain are subject to episodes of prolonged abnormal electrical activity. Spontaneous episodes of spreading depression (SD) have been observed in slices of rabbit hippocampus during a seizure-susceptible period that occurs at 8-12 days of age 5. During a similar period (age 9 - 1 9 days), the CA; region of the convulsant-treated rat hippocampus has been reported to display sustained periods of epileptiform activity, followed by prolonged afterdischarges 26. Developmental studies of epileptogenesis
in the rat neocortex have shown that several unique types of abnormal electrical activity occur in the immature brain3'4: spontaneous paroxysms of repetitive spike discharges, lasting as long as 30 s, occurred when slices from 8- to 15-day-old rats were exposed to convulsant drugs. Ion-sensitive microelectrodes have been widely used to document changes in extracellular ion concentrations during epileptiform discharges. Extracellular potassium ([K+]o) has repeatedly been shown to increase during interictal and ictal discharges in vivo 11'22'24 and in vitro 29, whereas extraceUular calcium ([Ca2+]o) decreases during such activity 7,8. Although changes in [Ca2+]o can precede the onset of paroxysmal activity 11, increases in [K+]o are generally viewed as being a result of the intense neuronal discharges associated with the abnormal activity, rather than a causal agent 2,17 in most but not all models of
* Present address: Institut ffir Normale und Pathologische Physiologie, Universit~it K61n, D-5000 K61n, F.R.G.
Correspondence: J.J. Hablitz, Section of Neurophysiology, Department of Neurology, Baylor College of Medicine, Houston, TX
77030, U.S.A.
0165-3806/87/$03.50© 1987 Elsevier Science Publishers B.V. (Biomedical Division)
300 epilepsy 16'29. Although the extent of [Ca2+],, decreases observed during epileptiform activity have been variable 2325, a ceiling level of 10-12 mM is generally observed for [K+]o increases 9,m with breaching of this ceiling generally resulting in the initiation of SD 21. However, [K+]o increases can be quite marked in the immature nervous system L2°,27,3°. suggesting an alteration in the [K+]o regulation mechanisms responsible for the usual [K+]o ceiling. [Ca2+],, decreases during epileptiform activity have not been extensively characterized in the developing brain. The present study demonstrates that ictal-like epileptiform activity in the immature rat neocortex is associated with decreases in [Ca2+]o similar to those observed in the adult neocortex. Abnormally large increases in [K+]o were observed, which exceeded the traditional ceiling level seen in the adult neocortex. Neocortical slices (400-500 # m thick) from 8- to 15-day-old rats were prepared and maintained in vitro as previously described 28. All slices were allowed to recover for at least 1 h before epileptiform activity was induced by bath application of picrotoxin (5 × 10 -5 M). In initial experiments, the reversibility of picrotoxin's effects was tested by washing with drugfree normal saline. Picrotoxin was present for the duration of the recording period in all other experiments. Recordings of extracellular ion concentrations and field potentials were made, using doublebarreled ion-sensitive/reference electrodes. ('a -'~and K+-sensitive electrodes were constructed and calibrated, using standard techniques Ij. For manufacture of the K+-sensitive electrodes, the C o m i n g 477317 or Fluka 60031 ion-exchange resin was used. The Fluka 21048 ion-sensitive cocktail was used for the Ca2+-sensitive electrodes, lonic changes and field potentials were typically measured 600 ~ m below the pial surface with the electrode inserted into the slice to a depth of 130-150/~m. Laminar profiles were constructed by moving the electrode to different depths below the pial surface and reinserting it to the same depth within the slice, A three-dimensional digital micropositioner was utilized to assure accurate positioning of the electrode. A chart recorder was used to make permanent records of ionic and fieldpotential changes. After exposure to picrotoxin, neocortical slices from 8- to 15-day-old rats generated spontaneous epileptiform activity. Extracellular field-potential
recordings revealed that this activity consisted of clusters of 3 - 5 discharges that resembled interictal spikes recorded in vivo Thesc clusters were interspersed with ictal-like paroxysms lasting 10-30 (Fig. 1A. lower trace). The epileptilorm activity reduced by picrotoxin, as well as the associated iomc changes described below, were no longer observed after washing with normal saline, Simultaneous measurement of field potentials and changes in [K~L showed that individual interictal spikes were associated with increases in [K+]o up l~, 12 raM. whereas increases to more than 20 mM accompanied longlasting ictal-like discharges (Fig. IA. uppm trace~ Examination of oscilloscope ~cc,,rdings indicated that [K+[o increases were very raDd Values of 10 mM could be reached within 600 ms. At the onset of ictal-like discharges, steplike increases m [K~],,, m-
A OOu,r
'-r
w
•
i
~
....
B IOOum
K
350
85(2
/
-
5f.,L ~
)
f100
los
600
Fig. 1, Recordings of [K+]~ changes and field potentials (Lp.) during spontaneous epileptiform activity m a neocortical slice from an tl-day-old animal. A: example ~f a recording made 100 .um below the cortical surface. Two types of epileptiform acnvity are seen. One consists of a group of 3-7 interictal spikes in the field-potential record, and the other is composed of an ictal-like discharge lasting tens of seconds. B: laminar analysis of changes in [K+lo and in field potentials. Largest [K+]o increases were observed at a depth of 600 ~m. This is also the depth at which the steady negative field potential was largest. Note the undershoot of [K+}~at this depth
301 tributable to individual interictal spikes, could be discerned on the rising phase of the potassium signal. These individual increments rapidly summated, and a plateau was maintained during the ictal-like event. Increases in [K÷]o were recorded throughout the cortex. Maximum increases were observed where the largest negative steady potentials were recorded by the field-potential electrode - - typically 600 ¢tm below the cortical surface (Fig. 1B). This depth is approximately layer V in this age group. When examined at this depth in 6 slices from 4 animals, [K+]o levels reached 20.1 + 5.7 mM during ictal-like discharges (n = 38). [K÷]o changes in the depths of the cortex were smaller in amplitude and slower in their rising and falling phases (Fig. 1B) compared with responses observed at more superficial recording sites. Interictal spikes and ictal-like discharges were associated with decreases in [Ca2+]o. As shown in Fig. 2A, individual interictal spikes produced [Ca2+]o decreases of 0.1-0.2 mM, whereas sustained discharges were accompanied by decreases of 0.3-0.4 mM. Lamina analysis of [Ca2+]o changes (Fig. 2B) indicated that the largest decreases occurred in superficial and middle layers of cortex, and smaller decreases were seen in deeper layers. The duration of the [Ca2+]o changes did not vary systematically as a function of depth below the cortical surface. Using ion-sensitive microelectrodes, we analyzed the changes in [K+]o and [Ca2+]o that occur in convulsant-treated neocortical slices from the immature brain. Our results indicate that the changes in [Ca2+]o were similar to those observed in mature animals, whereas the [K+]o accumulation was significantly higher than that seen in the adult brain during epileptiform discharges. The presently observed levels are similar to those observed in the immature hippocampus 27. The differences in [K+]o changes may account, in part, for the novel, ictal-like epileptiform activity seen in the 8- to 15-day-old animals. Paroxysmal discharges evoked by repetitive electrical stimulation of mature cat cortex in vivo produce small transient changes in [Ca2+]o (refs. 11, 25), whereas spontaneous seizure activity induced by pentylenetetrazol is associated with larger, sustained decreases in [Ca2+]o (ref. 8). In the convulsanttreated animals, decreases in [Ca2+]o were found in all cortical layers, whereas stimulus-induced changes were maximal in more superficial cortical layers 8.
A
450pm [Ca2+] o
-11.6mM 1.5 1.4
~
rF--m-7 JmV 1min
B 200}Jm
850
[c~]o,~-~~e, , ' f / ' i
] 1.6raM
"~F
t"s 1.4 1.3
450
1100 "~'~
77
--Ill-r-
--~,,~,,~
--"----
]2mV 10s
600
Fig. 2. Alterations in [Ca2+]o associated with picrotoxin-induced paroxysmal discharges in a slice from a 12-day-oldanimal. A: continuous recording of spontaneous interictal spiking and ictal-like discharges. Individual discharges produce detectable decreases in [Ca2+]o, whereas larger, more sustained decreases occur during the ictal-like event seen near the end of the record. B: specimen records of changes in field potentials (f.p.) and in [Ca2+]oat different depths below the cortical surface.
The changes in [Ca2+]o that we recorded in the neocortical slice preparation are similar to those observed in the intact neocortex. This suggests that although alterations in [Ca2+]o accompany epileptiform discharges in the 8- to 15-day-old group and may play a role in epileptogenesis, the changes observed in this age group do not differ qualitatively from those seen in the mature brain and are unlikely to be responsible for the unique types of abnormal activity observed. The mechanisms underlying the triggering of the spontaneous ictal-like discharges seen in the present
302 series of experiments are u n k n o w n . However. alterations in resting [K+]o, which occur in the immature brain in vivo 2°, do not appear to contribute to epileptogenesis in the slice preparation, since the resting [K+]o was stable at 5 mM throughout the experiments. Each interictal spike was associated with large increase in [K+]o, which rapidly s u m m a t e d during repetitive discharges. This sustained mcrease in [K+]o could heighten neuronal excitabilil y by directly depolarizing neurons, by reducing the action of repolarizing potassium currents, and by evoking the re~ lease of excitatory neurotransmitters, which suggests that elevation of [K÷]o mav be involved in the main-
optic nerve of the developing rat compared with adult values. Similarly, picrotoxin-induced p r o l o n g e d depolarizations in the developing cockroach have b e e n reported to be associated with [arge increases in [K +],, (ref. 30). As to the second possibility, in the rat neocortex, myelinization proceeds slowly in the postnatal period ~4. as does m a t u r a n o n of astrocyles ~-'~ The lack of myelin could result in an increased potassium release from an exposed population of axonat potassium channels al the same time that a full complement of mature astrocytes, necessary for potassium removal, is lacking. In the present study, m-
tenance of prolonged ictal-like epileptiform actiwt~
creases in [K+]o declined rapidly, and ictal-like discharges were followed by undershoots of the resting
in the immature neocortex.
potassium level This suggests the presence of an ac-
The cause of the higher levels of [K+]o associated
tive Na.K pump u in the immature neocortical slice
with repetitive firing in the immature neocortex is not yet known. Similar results have been obtained in the immature hippocampus -'7. which indicates that a n
preparation. However. significant increases in Na,K pump actiwty have been reported *o occur in the hippocampus during the 8- to 15-day postnatal period"
elevated ceiling for [K~],, may be a general propert3 of the neonatal period. Two possible mechanisms
If similar changes take place in the neocortex, a limited ability to clear potassium could be implicated in the high levels of [K+],, that we observed.
that could be involved in excessive accumulation ot potassium are (1) an e n h a n c e m e n t of factors responsible for potassium release, or (2} a diminished effectiveness of clearance processes
With regard to the
We would like to thank Dr. Jeffrey, Noebels for his
first mechanism, C o n n o r s et al.- reported that the rate of evoked potassium release is enhanced m the
helpful comments on an earlier version of this manuscript. This work was supported tn part by NIH Grants NS11535 and NS22373.
1 Connors, B.W., Ransom. B.R.. Kunis, D.M. and Gutnick. M.J., Activity-dependent K+ accumulation in the developing rat optic nerve, Science. 216 (19821 1341-1343. 2 Gutnick, M.J,, Heinemann. U. and Lux. H.D.. Stimulus induced and seizure related changes in extracellular potassium concentration in cat thalamus (VPL), Electroencephalogr. Clin. Neurophysiol., 47 (1979) 329-344. 3 Hablitz, J.J., Heinemann. U. and Weiss. D.S.. Epileptiform activity and spreading depression in the neocortical slice, Epilepsia, 25 (1984) 670. 4 Hablitz, J.J., Heinemann. U. and Weiss. D.S.. Cellular and ionic changes during epileptiform activity in the immature neocortex, Soc. Neurosci. Abstr. 12 (19861 727. 5 Haglund, M.M. and Schwartzkroin, P.A.. Seizure-like spreading depression in immature rabbit hippocampus in vitro, Dev. Brain Res.. 14 (1984) 51-59. 6 Haglund, M.M., Stahl. W.L.. Kunkel. D.D. and Schwartzkroin, P.A., Developmental and regional differences in the localization of Na.K-ATPase activity in the rabbit hippocampus, Brain Res.. 343 (1985) 198-203. 7 Heinemann, U., Konnerth, A and Lux. H.D.. Stimulation induced changes in extracellular free calcium in normal cortex and chronic alumina cream foci of cats. Brain Res.. 213 (1981) 246-250. 8 Heinemann, U. and Louvel. J.. Changes in [Ca>L, and
[K + ]<, during rcpentive electrical stimulation and during pentetrazol induced seizure activity in the sensorimotor cortex of cats. Pflugers Arch., 398 f1983}310-317 Heinemann. U. and Lux. H.D. I Jndershoots following stimulus induced rises of extracellular potassium concentration in cerebral cortex of cat. Brain Re.~.. 93 11975) 63--76 10 Heinemann. U. and Lux. H.D., Ceiling ot"stimulus induced rises in extracellular potassium concentration in the cerebral cortex of cats. Brain Res., 12tlt t977) 231-249. I1 Heincmann. U. Lux. H.D. and Gulnick. M.J.. ExtracctluJar free calcium and potassium during paroxysmal activity in cerebral cortex of the cat. Ext Rrain Res. 27 flq771 237-243, 12 Holtzman. D.. Obana. K, and Olson. J., Hyperthermia-induced seizures in the rat pup: a model for febrile convulsions in children. Science. 213 fI981) 1034-1036. 13 Ichikawa. M. and Hirata. Y.. Morphology and distribution of postnatally generated glial cells in the somatosensory cortex of the rat: an autoradiographic and electron microscopic study, Brain Res.. 4 (1982) 369-377. 14 Jacobson, S.. Sequence of myelinization m the brain of the albino rat. A Cerebral cortex, thalamus and related structures. J. Comp. Neurol., 12 ( 1963] 5-29. 15 Kellaway. P. and Hrachovy_ R.A., Status epilepticus m newborns: a perspective on neonatal smzures tn A.V. Del-
303
16
17
18
19
20 21
22
23
gado-Escueta, C.G. Wasterlain, D.M. Treiman and R.J. Porter (Eds.), Status Epilepticus: Mechanisms of Brain Damage and Treatment, Advances in Neurology, Vol. 34, Raven, New York, 1983, pp. 93-99. Konnerth, A., Heinemann, U. and Yaari, Y., Slow transmission of neural activity in hippocampal area CA t in absence of active chemical synapses, Nature (London), 307 (1984) 69-71. Moody, W.J., Jr., Futamachi, K.J. and Prince, D.A., Extracellular potassium activity during epileptogenesis, Exp. Neurol., 42 (1974) 248-263. Moshe, S.L. and Albala, B.J., Maturational changes in postictal refractoriness and seizure susceptibility in developing rats, Ann. Neurol., 13 (1983) 552-557. Moshe, S.L., Albala, B.J., Ackermann, R.F. and Engel, Jr., J., Increased seizure susceptibility of the immature brain, Dev. Brain Res., 7 (1983) 81-85. Mutani, R., Futamachi, K.J. and Prince, D.A., Potassium activity in immature cortex, Brain Res., 75 (1974) 27-39. Nicholson, C. and Kraig, R.P., The behavior of extracellular ions during spreading depression. In T. Zeuthen (Ed.), The Application of Ion-Selective Microelectrodes, Elsevier, Amsterdam, 1981, pp. 217-238. Prince, D.A., Neher, E. and Lux, H.D., Measurement of extracellular potassium activity in cat cortex, Brain Res., 50 (1973) 489-495. Pumain, R., Meini, C., Heinemann, U., Louvel, J. and Silva-Barrat, C., Chemical synaptic transmission is not neces-
sary for epileptic seizures to persist in the baboon, Papio papio, Exp. Neurol., 89 (1985) 250-258. 24 Somjen, G.G., Extracellular potassium in the mammalian central nervous system, Annu. Rev. Physiol., 41 (1979) 159-177. 25 Somjen, G.G., Stimulus-evoked and seizure-related responses of extracellular calcium activity in spinal cord compared to those in cerebral cortex, J. Neurophysiol., 44 (1980) 617-632. 26 Swann, J.W. and Brady. R.J., Penicillin-induced epileptogenesis in immature rat CA~ hippocampal pyramidal cells, Dev. Brain Res., 12 (1984) 243-254. 27 Swarm, J.W., Smith, K.L. and Brady, R.J., Extracellular K + accumulation during penicillin-induced epileptogenesis in the CA 3 region of immature rat hippocampus, Dev. Brain Res., 30 (1986) 243-255. 28 Weiss, D.S. and Hablitz, J.J., Interaction of penicillin and pentobarbital with inhibitory synaptic mechanisms in neocortex, Cell. Mol. Neurobiol.. 4 (1984) 3{)1-317. 29 Yaari, Y., Konnerth, A. and Heinemann, U., Nonsynaptic epileptogenesis in the mammalian hippocampus in vitro. II. Role of extracellular potassium, J. Neurophysiol., 56 (1986) 424-438. 30 Yarom, Y., Grossman, Y., Gutnick, M.J. and Spira, M.E., Transient extracellular potassium accumulation produced prolonged depolarizations during synchronized bursts in picrotoxin-treated cockraoch CNS, J. Neurophysiol., 48 (1982) 1089-1097.
299
Elsevier BRD 60239
Extracellular K + and Ca 2+ changes during epileptiform discharges in the immature rat neocortex John J. Hablitz and Uwe Heinemann* Section of Neurophysiology, Departmentof Neurology, Baylor Collegeof Medicine, Houston, Texas 77030(U.S.A.) and Department of Neurophysiology, Max-PlanckInstitutefor Psychiatry, Planegg-Martinsried(F.R.G.) (Accepted 18 August 1987)
Key words: Neocortex; Epileptiform activity; Extracellular potassium; Extracellular calcium; Development
Picrotoxin-induced epileptiform activity was examined in neocortical slices prepared from 8- to 15-day-old rats. This activity consisted of spontaneous bursts of 3-5 discharges that resembled interictal spikes and were interspersed with ictal-like paroxysms lasting 10-30 s. Measurements of extracellular potassium ([K+]o) and calcium ([Ca2+]o)were made during these spontaneous epileptiform events, using ion-sensitive electrodes. Individual interictal spikes were associated with [Ca2+]odecreases of 0,1-0.2 mM, whereas sustained ictal-like discharges were accompanied by decreases of 0.3-0.4 mM. Measurement of [K+]o showed that individual interictal spikes were associated with increases in [K+]oup to t2 raM, whereas increases to more than 20 mM accompanied long-lasting ictal-like discharges. Maximum increases in [K+]o were observed ca. 600 pm below the pial surface. [K+]o increases were followed by undershoots of the resting [K÷]o level. The unusually high [K÷]o levels associated with epileptiform discharges in the immature neocortex suggest that disturbances in [K÷]o regulation may contribute to the generation of the picrotoxin-induced, spontaneous, prolonged ictal-like discharges observed in the 8- to 15-day age group.
The incidence of seizure disorders in early life is quite high in man, and several unique forms of epilepsy occur in the neonatal period iS. Experimental studies in neonatal rats have demonstrated that the brain is more prone to manifest epileptiform discharges at certain times during development j2' Is~'). Studies in vitro have documented that during postnatal development, there is a stage in which certain regions of the brain are subject to episodes of prolonged abnormal electrical activity. Spontaneous episodes of spreading depression (SD) have been observed in slices of rabbit hippocampus during a seizure-susceptible period that occurs at 8-12 days of age 5. During a similar period (age 9 - 1 9 days), the CA; region of the convulsant-treated rat hippocampus has been reported to display sustained periods of epileptiform activity, followed by prolonged afterdischarges 26. Developmental studies of epileptogenesis
in the rat neocortex have shown that several unique types of abnormal electrical activity occur in the immature brain3'4: spontaneous paroxysms of repetitive spike discharges, lasting as long as 30 s, occurred when slices from 8- to 15-day-old rats were exposed to convulsant drugs. Ion-sensitive microelectrodes have been widely used to document changes in extracellular ion concentrations during epileptiform discharges. Extracellular potassium ([K+]o) has repeatedly been shown to increase during interictal and ictal discharges in vivo 11'22'24 and in vitro 29, whereas extraceUular calcium ([Ca2+]o) decreases during such activity 7,8. Although changes in [Ca2+]o can precede the onset of paroxysmal activity 11, increases in [K+]o are generally viewed as being a result of the intense neuronal discharges associated with the abnormal activity, rather than a causal agent 2,17 in most but not all models of
* Present address: Institut ffir Normale und Pathologische Physiologie, Universit~it K61n, D-5000 K61n, F.R.G.
Correspondence: J.J. Hablitz, Section of Neurophysiology, Department of Neurology, Baylor College of Medicine, Houston, TX
77030, U.S.A.
0165-3806/87/$03.50© 1987 Elsevier Science Publishers B.V. (Biomedical Division)
300 epilepsy 16'29. Although the extent of [Ca2+],, decreases observed during epileptiform activity have been variable 2325, a ceiling level of 10-12 mM is generally observed for [K+]o increases 9,m with breaching of this ceiling generally resulting in the initiation of SD 21. However, [K+]o increases can be quite marked in the immature nervous system L2°,27,3°. suggesting an alteration in the [K+]o regulation mechanisms responsible for the usual [K+]o ceiling. [Ca2+],, decreases during epileptiform activity have not been extensively characterized in the developing brain. The present study demonstrates that ictal-like epileptiform activity in the immature rat neocortex is associated with decreases in [Ca2+]o similar to those observed in the adult neocortex. Abnormally large increases in [K+]o were observed, which exceeded the traditional ceiling level seen in the adult neocortex. Neocortical slices (400-500 # m thick) from 8- to 15-day-old rats were prepared and maintained in vitro as previously described 28. All slices were allowed to recover for at least 1 h before epileptiform activity was induced by bath application of picrotoxin (5 × 10 -5 M). In initial experiments, the reversibility of picrotoxin's effects was tested by washing with drugfree normal saline. Picrotoxin was present for the duration of the recording period in all other experiments. Recordings of extracellular ion concentrations and field potentials were made, using doublebarreled ion-sensitive/reference electrodes. ('a -'~and K+-sensitive electrodes were constructed and calibrated, using standard techniques Ij. For manufacture of the K+-sensitive electrodes, the C o m i n g 477317 or Fluka 60031 ion-exchange resin was used. The Fluka 21048 ion-sensitive cocktail was used for the Ca2+-sensitive electrodes, lonic changes and field potentials were typically measured 600 ~ m below the pial surface with the electrode inserted into the slice to a depth of 130-150/~m. Laminar profiles were constructed by moving the electrode to different depths below the pial surface and reinserting it to the same depth within the slice, A three-dimensional digital micropositioner was utilized to assure accurate positioning of the electrode. A chart recorder was used to make permanent records of ionic and fieldpotential changes. After exposure to picrotoxin, neocortical slices from 8- to 15-day-old rats generated spontaneous epileptiform activity. Extracellular field-potential
recordings revealed that this activity consisted of clusters of 3 - 5 discharges that resembled interictal spikes recorded in vivo Thesc clusters were interspersed with ictal-like paroxysms lasting 10-30 (Fig. 1A. lower trace). The epileptilorm activity reduced by picrotoxin, as well as the associated iomc changes described below, were no longer observed after washing with normal saline, Simultaneous measurement of field potentials and changes in [K~L showed that individual interictal spikes were associated with increases in [K+]o up l~, 12 raM. whereas increases to more than 20 mM accompanied longlasting ictal-like discharges (Fig. IA. uppm trace~ Examination of oscilloscope ~cc,,rdings indicated that [K+[o increases were very raDd Values of 10 mM could be reached within 600 ms. At the onset of ictal-like discharges, steplike increases m [K~],,, m-
A OOu,r
'-r
w
•
i
~
....
B IOOum
K
350
85(2
/
-
5f.,L ~
)
f100
los
600
Fig. 1, Recordings of [K+]~ changes and field potentials (Lp.) during spontaneous epileptiform activity m a neocortical slice from an tl-day-old animal. A: example ~f a recording made 100 .um below the cortical surface. Two types of epileptiform acnvity are seen. One consists of a group of 3-7 interictal spikes in the field-potential record, and the other is composed of an ictal-like discharge lasting tens of seconds. B: laminar analysis of changes in [K+lo and in field potentials. Largest [K+]o increases were observed at a depth of 600 ~m. This is also the depth at which the steady negative field potential was largest. Note the undershoot of [K+}~at this depth
301 tributable to individual interictal spikes, could be discerned on the rising phase of the potassium signal. These individual increments rapidly summated, and a plateau was maintained during the ictal-like event. Increases in [K÷]o were recorded throughout the cortex. Maximum increases were observed where the largest negative steady potentials were recorded by the field-potential electrode - - typically 600 ¢tm below the cortical surface (Fig. 1B). This depth is approximately layer V in this age group. When examined at this depth in 6 slices from 4 animals, [K+]o levels reached 20.1 + 5.7 mM during ictal-like discharges (n = 38). [K÷]o changes in the depths of the cortex were smaller in amplitude and slower in their rising and falling phases (Fig. 1B) compared with responses observed at more superficial recording sites. Interictal spikes and ictal-like discharges were associated with decreases in [Ca2+]o. As shown in Fig. 2A, individual interictal spikes produced [Ca2+]o decreases of 0.1-0.2 mM, whereas sustained discharges were accompanied by decreases of 0.3-0.4 mM. Lamina analysis of [Ca2+]o changes (Fig. 2B) indicated that the largest decreases occurred in superficial and middle layers of cortex, and smaller decreases were seen in deeper layers. The duration of the [Ca2+]o changes did not vary systematically as a function of depth below the cortical surface. Using ion-sensitive microelectrodes, we analyzed the changes in [K+]o and [Ca2+]o that occur in convulsant-treated neocortical slices from the immature brain. Our results indicate that the changes in [Ca2+]o were similar to those observed in mature animals, whereas the [K+]o accumulation was significantly higher than that seen in the adult brain during epileptiform discharges. The presently observed levels are similar to those observed in the immature hippocampus 27. The differences in [K+]o changes may account, in part, for the novel, ictal-like epileptiform activity seen in the 8- to 15-day-old animals. Paroxysmal discharges evoked by repetitive electrical stimulation of mature cat cortex in vivo produce small transient changes in [Ca2+]o (refs. 11, 25), whereas spontaneous seizure activity induced by pentylenetetrazol is associated with larger, sustained decreases in [Ca2+]o (ref. 8). In the convulsanttreated animals, decreases in [Ca2+]o were found in all cortical layers, whereas stimulus-induced changes were maximal in more superficial cortical layers 8.
A
450pm [Ca2+] o
-11.6mM 1.5 1.4
~
rF--m-7 JmV 1min
B 200}Jm
850
[c~]o,~-~~e, , ' f / ' i
] 1.6raM
"~F
t"s 1.4 1.3
450
1100 "~'~
77
--Ill-r-
--~,,~,,~
--"----
]2mV 10s
600
Fig. 2. Alterations in [Ca2+]o associated with picrotoxin-induced paroxysmal discharges in a slice from a 12-day-oldanimal. A: continuous recording of spontaneous interictal spiking and ictal-like discharges. Individual discharges produce detectable decreases in [Ca2+]o, whereas larger, more sustained decreases occur during the ictal-like event seen near the end of the record. B: specimen records of changes in field potentials (f.p.) and in [Ca2+]oat different depths below the cortical surface.
The changes in [Ca2+]o that we recorded in the neocortical slice preparation are similar to those observed in the intact neocortex. This suggests that although alterations in [Ca2+]o accompany epileptiform discharges in the 8- to 15-day-old group and may play a role in epileptogenesis, the changes observed in this age group do not differ qualitatively from those seen in the mature brain and are unlikely to be responsible for the unique types of abnormal activity observed. The mechanisms underlying the triggering of the spontaneous ictal-like discharges seen in the present
302 series of experiments are u n k n o w n . However. alterations in resting [K+]o, which occur in the immature brain in vivo 2°, do not appear to contribute to epileptogenesis in the slice preparation, since the resting [K+]o was stable at 5 mM throughout the experiments. Each interictal spike was associated with large increase in [K+]o, which rapidly s u m m a t e d during repetitive discharges. This sustained mcrease in [K+]o could heighten neuronal excitabilil y by directly depolarizing neurons, by reducing the action of repolarizing potassium currents, and by evoking the re~ lease of excitatory neurotransmitters, which suggests that elevation of [K÷]o mav be involved in the main-
optic nerve of the developing rat compared with adult values. Similarly, picrotoxin-induced p r o l o n g e d depolarizations in the developing cockroach have b e e n reported to be associated with [arge increases in [K +],, (ref. 30). As to the second possibility, in the rat neocortex, myelinization proceeds slowly in the postnatal period ~4. as does m a t u r a n o n of astrocyles ~-'~ The lack of myelin could result in an increased potassium release from an exposed population of axonat potassium channels al the same time that a full complement of mature astrocytes, necessary for potassium removal, is lacking. In the present study, m-
tenance of prolonged ictal-like epileptiform actiwt~
creases in [K+]o declined rapidly, and ictal-like discharges were followed by undershoots of the resting
in the immature neocortex.
potassium level This suggests the presence of an ac-
The cause of the higher levels of [K+]o associated
tive Na.K pump u in the immature neocortical slice
with repetitive firing in the immature neocortex is not yet known. Similar results have been obtained in the immature hippocampus -'7. which indicates that a n
preparation. However. significant increases in Na,K pump actiwty have been reported *o occur in the hippocampus during the 8- to 15-day postnatal period"
elevated ceiling for [K~],, may be a general propert3 of the neonatal period. Two possible mechanisms
If similar changes take place in the neocortex, a limited ability to clear potassium could be implicated in the high levels of [K+],, that we observed.
that could be involved in excessive accumulation ot potassium are (1) an e n h a n c e m e n t of factors responsible for potassium release, or (2} a diminished effectiveness of clearance processes
With regard to the
We would like to thank Dr. Jeffrey, Noebels for his
first mechanism, C o n n o r s et al.- reported that the rate of evoked potassium release is enhanced m the
helpful comments on an earlier version of this manuscript. This work was supported tn part by NIH Grants NS11535 and NS22373.
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