Brain Research 885 2000 102–110 www.elsevier.com locate bres
Research report
Potassium-induced enhancement of persistent inward current in hippocampal neurons in isolation and in tissue slices
1
¨ G.G. Somjen , M. Muller
Department of Cell Biology , Box 3709, Duke University Medical Center, Durham, NC 27710, USA
Received 19 May 2000; accepted 12 September 2000
Abstract
Previous work suggested a role for the voltage-dependent persistent sodium current, I , in the generation of seizures and spreading
Na,P 1
depression SD. Ordinarily, I is small in hippocampal neurons. We investigated the effect of raising external K
concentration,
Na,P 1
[K ] , on whole-cell persistent inward current in freshly isolated hippocampal CA1 pyramidal neurons. I was identified by
o Na,P
1
TTX-sensitivity and dependence on external Na concentration. When none of the ion channels were blocked, I
was not usually
Na,P 1
1 1
detectable, probably because competing K current masked it, but after raising [K ] I
appeared, while K currents diminished. With
o Na,P
1 1
K channels blocked, I
could usually be evoked in control solution and raising [K ] caused its reversible increase in most cells. The
Na,P o
21
increase did not depend on external calcium [Ca ] . In CA1 pyramidal neurons in hippocampal slices a TTX-sensitive persistent inward
o 1
current was always recorded and when [K ] was raised, it was reversibly enhanced. Strong depolarization evoked irregular current
o 1
fluctuations, which were also augmented in high [K ] . The findings support a role of potassium-mediated positive feedback in the
o
generation of seizures and spreading depression.
2000 Elsevier Science B.V. All rights reserved.
Theme : Excitable membranes and synaptic transmission
Topic : Sodium channels
Keywords : Persistent sodium current; Sodium channel; External potassium; Intracellular calcium; Seizure; Spreading depression
1. Introduction component of the extracellular potential shift during ictal
seizures revealed a long-lasting prominent current sink
1
Accumulation of K ions in cerebral interstitial spaces
limited to cell body layers, for which the source was has been blamed for the eruption of spreading depression
distributed over the dendritic fields [45]. By contrast,
1
SD by Grafstein [17] and for the generation of seizure voltage shifts and [K ] increase associated with SD start
o
discharges by Green [18] and by Fertziger and Ranck [12]. in the dendrite layers [24] where intrinsic optical signals
Gloor et al. [15,16] identified a sustained extracellular are also maximal [1,34] and CSD shows powerful inward
negative potential shift during epileptiform seizures that currents [45]. In spite of the obvious differences between
was limited to the cell body layers in dentate gyrus and seizures and SD, there are also important similarities. Both,
hippocampus. We have confirmed this observation [40] and seizures and SD can be triggered by similar insults, and
1
demonstrated that the accumulation of K ions was also
both are inhibited by similar physical or pharmacological maximal in the interstitium of cell body layers [41].
interventions, such as cooling, hypertonicity, acidosis and Current source density CSD analysis of the sustained
certain depressant drugs [4,32]. An event that begins with an epileptiform discharge can sometimes terminate in SD
[39] and a seizure that starts in a focus can then spread
Corresponding author. Tel.: 11-919-681-8404; fax: 11-919-684-
over a large area with a velocity resembling that of SD
5481.
[4,44].
E-mail address : g.somjencellbio.duke.edu G.G. Somjen.
1
From these and similar observations it has been clear
¨ Present address: Zentrum fur Physiologie und Pathophysiologie, Ab-
that both tonic seizures and spreading depression require
¨ teilung Neuro- und Sinnesphysiologie Humboldtallee 23, D-37073 Gott-
ingen, Germany.
inward currents that inactivate very slowly or not at all.
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 4 8 - 6
G .G. Somjen, M. Muller Brain Research 885 2000 102 –110
103
The nature of these currents was, however not known. A microscope objective with patch pipettes; tight seal was
voltage-dependent persistent sodium current was demon- established, and the whole-cell recording condition created
strated, amongst others, in hippocampal neurons [13,26] by suction. To record Na as well as K currents the pipettes
reviewed by Crill [7] and by Taylor [43]. It is not known were filled with a solution containing in mmol l: KF
1
whether I flows through distinct classes of Na
chan- 129, NaCl 4, EGTA 10, CaCl 0.5, MgCl 2, Hepes 10,
Na,P 2
2
nels, or whether it represents failure of inactivation of the Na ATP 4, pH 7.1 or 7.3, tip resistance 2.5–4.5 MV. To
2 1
1
‘conventional’, fast, transient Na current. Recently the
block K currents, KF was substituted by 109 mM CsF
weight of evidence seemed to be shifting toward the latter and 20 mM TEA-Cl.
interpretation [2,31]. An Axopatch 1D amplifier in voltage clamp configura-
The physiological role attributed to I is the regula-
tion and the pClamp-6 Axon Instruments suite of pro-
Na,P
tion of the ‘resting’ excitability of neurons. Its usual grams was used to record whole-cell currents. Pipette and
conductance is too weak to explain the depolarization seen cell capacitances were compensated in the customary
in such severely pathological conditions as seizures or SD. manner. Series resistance was compensated to 70. The
I greatly increases, however, under pathological con-
holding potential was 270 mV pipette voltage. Current–
Na,P 1
ditions. In heart and skeletal muscle a persistent Na voltage I –V curves were recorded usually at one, some-
1
current is enhanced by elevated [K ] [9] and hypoxia
times at 2-min intervals. Two different protocols were
o
causes an increase in heart muscle [5,28] as well as in used: either eight sweeps, each beginning with a pre-pulse
neurons [19]. In computer-based simulations we recently of 100 ms to 290 mV to remove inactivation, followed by
1
demonstrated that Na -mediated, voltage-dependent, slow- 200-ms depolarizing steps at 2-s intervals in 15-mV
ly inactivating inward current can generate an SD-like increments, taking the pipette voltage from 270 to 135
depolarized state [29]. We have therefore asked whether mV; or 12 sweeps of a 200-ms hyperpolarizing pre-pulse
1
elevation of [K ] would also cause an increase in I in
followed by 400-ms depolarizing steps in 10-mV incre-
o Na,P
hippocampal neurons. This indeed turned out to be the ments, taking the pipette from 270 to 140 mV.
case. Some of the findings have been reported in an The current records were read with Clampfit Axon
abstract [38]. Instruments software. After subtraction of linear leak and
holding currents, the data were further processed with the Excel Microsoft program. Junction potentials were calcu-
2. Methods lated with the JPCalc program [3].
