Results high [K ] solution.
104 G
Slices were left undisturbed for 90 min before recording slowly inactivating inward current was detectable in all but
began. two of a population of 42 isolated CA1 hippocampal
neurons. The mean amplitude normalized to cell capaci- 2.5. Whole-cell recording in tissue slices
tance was 27.166.5 pA pF mean6st.d., with a range from 0 to 224.3 pA pF in cells with average capacitance
1
Patch pipettes were pulled from thick-walled glass tubes of 10.762.2 pF. In 10 trials the bath K
concentration was and filled with the following solution in mmol l: 120
raised from 3 to 20 mM; the mean persistent inward K-gluconate, 8 KCl, 10 Hepes, 11 EGTA, 0.5 CaCl , 2
current in this sample in control solution was 24.860.9
2
MgCl , 4 ATP-Na , pH 7.35, osmolarity 290, pipette tip pA pF mean6S.E.M. and it increased to 28.461.6 pA
2 2
resistance 3–5 MV. Cells in CA1 stratum pyramidale were pF P ,0.03 by paired t-test. Fig. 1 illustrates an example
found by ‘blind’ search. Seal resistances of 0.5 GV, of the reversible enhancement of the persistent inward
more usually 1.0 GV were accepted. Pipette capacitance current in one of these cells. In this case the maximal
was compensated. Whole-cell recording condition was persistent inward current increased during elevation of
1
established by gentle suction. Cell capacitance and series [K ]
by a factor of almost 4 Fig. 1A,C while the
o
resistance were not compensated. Holding potential was calculated maximal conductance doubled Fig. 1D. The
265 mV. Series of test pulses were delivered at 90-s sample currents of Fig. 1B show an increase not only of
intervals. Each protocol consisted of a pre-pulse of 400 ms the slowly inactivating inward current, but also of an
to 290 mV followed by a series of either eight or 10 inward tail current that follows repolarization. As long as
depolarizing steps of 600 ms in 10-mV increments, either the persistent inward current was small, tail currents were
from 270 to 0, or from 270 to 120 mV. Data processing usually small or absent in recordings made with CsF
was similar to that for isolated cells. pipettes. Marked tail currents were seen whenever the
Each cell was examined first for 15 min in normal persistent inward current grew large, but there was no
1
solution, then for 15 min in elevated K 10 mmol l
simple correlation between the amplitudes of the two. solution. If the seal held, then either recovery was ob-
In the absence of channel blocking agents, when KF- served during washing with normal ACSF for 15–30 min,
filled pipettes were used, the persistent current was a
1 1
or the cell was exposed to high K solution with tet-
mixture mainly composed of the delayed rectifier K rodotoxin TTX 1.0 mM added. Slices were exposed to
current, I [36] and I
. As long as the cells were
K,DR Na,P
1
high K only once.
bathed in normal solution, a persistent inward current was
1
detected in only two out of 11 cells. When the K 2.6. Statistics
concentration in the bath was raised, such an inward current appeared in nine of the 11 cells. Its amplitude was
Except when otherwise noted, numerical data are given 21.1 pA pF n53 at 10 mM, 23.6 pA pF n57 at 20
1
as the mean6S.E.M. Significance was calculated by paired mM and 216.9 pA pF n 53 at 40 mM [K ] . Apparent-
o
two-tailed t-test. ly in control solution the powerful I
masked the weak
K,DR 1
I , but as [K ] increased, I
strengthened sufficiently
Na,P o
Na,P
to compete. Fig. 2B illustrates the emergence of I in
Na,P 1
3. Results high [K ] solution.
o 1
The transient Na current, I
, usually increased in the
Na,T
3.1. The effect of elevated potassium concentration, first few minutes of recording from a cell, and then its
1
[K ] , on isolated neurons amplitude drifted slowly over time, either increasing or
o 1
decreasing. Raising [K ] did not alter this course. In Fig.
o
Whole-cell currents were evoked by depolarizing volt- 2 the I –V curves and the normalized conductances of I
Na,T
age steps in patch-clamped neurons freshly isolated from and I
of one cell are compared.
Na,P
CA1 region of rat hippocampus. The recording pipettes were filled either with a KF-based solution that does not
3.2. Elevated potassium concentration and fluorescence impede any of the known major ion currents, or a solution
1
with CsF as the major electrolyte and 20 mM TEA , to Laser-induced fluorescence in cells filled with calcium
1
block most K -mediated outward currents. The holding indicator dyes causes a powerful, slowly reversing poten-
potential was 270 mV. Pipette and cell capacitances were tiation of I
[37,38]. To see whether the effects of
Na,P 1
neutralized and series resistance was 70 compensated. elevated [K ] and of fluorescence interact, dye-filled cells
o
Linear leak and holding current were subtracted off line. were intermittently exposed to laser light while voltage-
To construct current–voltage I –V curves, cells were dependent currents were also tested, first in normal solu-
1
stimulated by series of depolarizing steps, preceded by tion and then in elevated [K ] . Currents were recorded
o
hyperpolarization to 290 mV. Sets of test pulses were with KF filled micropipettes, so that the persistent inward
applied at 1- or 2-min intervals. currents were, initially, quite small or absent. Fig. 3
When K-currents were blocked, a voltage dependent, illustrates the changes of the maximal persistent inward
G .G. Somjen, M. Muller Brain Research 885 2000 102 –110
105
Fig. 1. Reversible enhancement of persistent inward sodium current, I in an isolated neuron during exposure to elevated external potassium
Na,P 1
1
concentration. A–D Data from the same cell, recorded with a pipette filled with a solution based on CsF, containing 20 mM TEA , to block most K currents. No fluorescent dyes were used. Current amplitudes for A, C and D were measured as the average during the last 15 ms of depolarization. A
1
The maximal amplitude of the persistent inward current recorded before, during and after exposure to 20 mM [K ] in the bath. The broken vertical lines indicate change of bath solution. The horizontal bar indicates intermittent illumination by laser but, in the absence of dyes, the illumination had no effect on
I . B Sample recordings of currents evoked by voltage step to 210 mV pipette potential, corresponding to membrane potential V of 216.7 mV after
Na,P m
1 1
correction for junction potential. Superimposed recordings obtained before raising [K ] , 8 min after switching to high K solution, and after 20 min of
o
washing with normal solution. Sampling rate 2000 Hz, filtered off-line at 100 Hz. C Current–voltage I –V curves of the slowly inactivating current
1 1
obtained just before switching to high K solution, after 8 min of exposure to 20 mM [K ] , after 8 min of intermittent laser illumination while exposed to
o 1
high [K ] , and after 20 min washing with normal ACSF. D Whole-cell conductance in nanoSiemens as function of V , calculated by dividing the
o m
currents shown in part C by the driving potential, defined as the difference between V and the reversal potential of the current.
m
21
currents recorded at one min intervals from 10 such dye- of influx of Ca
through voltage-dependent channels. These increases dissipated slowly, creating a ‘sawtooth’
filled cells. The rapid increase of the inward current after
1
pattern when the fluorescence ratio was plotted against high-K
solution was introduced into the bath, strongly
1
time Fig. 4A. The amplitude of these voltage-dependent suggests that raising [K ]
accelerated the laser-induced
o 21
1
Ca responses gradually decreased with repetition, proba-
increase. Of the 10 cells three were lost during the high K
1
bly due to rundown. Raising [K ] caused a small increase treatment. When the seven remaining cells were washed in
o
in the baseline fluorescence ratio in most cells, indicating normal solution, the inward current decreased for a short
21
an increase in ‘resting’ [Ca ] . The magnitude of the
period, but in five of the cells it then resumed to increase
i
enhancement of I and of the change in fluorescence
under the influence of the continuing laser pulses.
Na,P
ratio baseline were, however, not correlated. Laser illumination of cells that were not filled with dye
The effect of calcium on I was further tested in three
had no effect on the persistent inward current, regardless of
Na,P 21
1 1
cells by removing Ca from the bath while raising [K ].
whether the bath K concentration was normal, or elevated
21 21
Fig. 1. Lack of [Ca
] was compensated by raising Mg
o
concentration from 1.0 to 2.2 mM. Fig. 4B,C illustrates
1
3.3. K -induced augmentation of I is not calcium
one such trial, recorded with a CsF-filled pipette. As
Na ,P
21
dependent expected, removing Ca
suppressed the ‘sawtooth’ oscil- lations and depressed the baseline fluorescence Fig. 4A.
21
The depolarizing pulses that were applied to obtain I –V Mobilization of Ca
from internal stores, if any, could not curves caused the fluorescence ratio to increase, as a result
compensate for the reduction of the external source of the
106 G
1 1
N-methyl-
D
-glucamine NMDG for 50 mM Na in the
1
bath, in the presence of 20 mM [K ] in four fluorescent
o
cells, depressed I from 2117639 to 238610 pA pF
Na,P 1
P ,0.04. TTX-sensitivity, reversal potential, and Na dependence confirmed that this is a slowly inactivating
sodium current, I .
Na,P
3.5. Neurons in tissue slices Since isolated neurons are truncated and are in an
1
abnormal environment, we asked whether raising [K ]
o
would also enhance I in intact neurons in their natural
Na,P
habitat. To answer this question, whole-cell recordings were made from pyramidal cells in CA1 region of rat
hippocampal tissue slices. The slices were maintained in an interface tissue slice chamber perfused with bicar-
bonate-buffered artificial cerebrospinal fluid ACSF at 368C. Glucose in the bath was 10 mM, instead of the 25
mM used for isolated cells. The pipettes were filled with K-gluconate-based solution and no ion channel blocking
drugs were used, except at the end of some trials when 1
1
mM TTX was added to the bath to verify the Na dependence of the inward currents.
The voltage of cells in situ cannot perfectly be con- trolled by the whole-cell patch clamp method, because the
membrane potential of the axon and of the dendritic tree cannot be controlled by feedback applied to the cell body.
Nonetheless, with this technique changes in the relative magnitude of the voltage-induced currents can reliably be
assessed. Since the membrane potential was presumably not uniform over the entire cell surface, in this section we
report pipette voltages without correction for series resist- ance or junction potential, because this was the only
reliably known voltage. The capacitance of the cells in tissue was not neutralized, but this does not introduce a
serious error in the measurement of currents that change
Fig. 2. Comparing the persistent and the transient sodium currents. From
only slowly. The holding potential was 265 mV; to obtain
one cell, recorded by KF filled micropipette. A I –V curves of the
I –V curves the pipette was first stepped to 290 mV for 400
maximal amplitude of the fast transient inward current, I , in control
Na,T 1
ms and then a series of 600-ms depolarizing pulses were
solution and in 20 mM bath [K ]. B I –V curves of the persistent current
applied in 10-mV increments, either from 270 to 0 mV or
measured during the last 15 ms of depolarization from the same traces as those used for A. In the absence of channel blocking drugs the persistent
from 270 to 120 mV. Depolarization evoked trains of
current is a mixture consisting mainly of I and I
. C Voltage
Na,P K,DR
current spikes presumably drawn by action potentials fired
dependence of the normalized conductances of I and of the mixed
Na,T
in the axon, outside the voltage clamped region, as well as
1
persistent current in elevated [K ] . Conductance was calculated by
o
slower transient inward current surges probably reflecting
dividing current by driving voltage, normalized to the maximal amplitude.
calcium action potentials generated in the dendritic tree [8,27]. Persistent currents were measured within segments
1
ion. During the same bath change, [K ] was raised to 10 of traces toward the end of the depolarizing voltage steps
o
mM. I was augmented, demonstrating the independence
where firing ceased, or else between spikes.
Na,P 1
21
of the high [K ] effect from Ca influx Fig. 4B.
After subtracting linear ‘leak’ and holding current off
o
line, a slowly inactivating inward current was evident in
1 1
3.4. TTX and low [Na ] suppress high K -augmented recordings from all observed cells in normal solution Figs.
o
persistent inward current 5 and 6 also noted in an earlier study, see Ref. [27]. I –V
curves were recorded at 90-s intervals over a period of Exposing cells to 0.5 mM tetrodotoxin TTX sup-
15–16.5 min in control solution, followed by a similar
1
pressed the laser- and high [K ] -enhanced persistent length of time during perfusion with solution containing 10
o 1
inward current n 53 see also Ref. [37]. Substituting mM K . Equilibration of ion concentrations between the
G .G. Somjen, M. Muller Brain Research 885 2000 102 –110
107
1
Fig. 3. Raising [K ] hastens fluorescence-induced potentiation of I . The maximal persistent inward currents measured in 10 fluorescent isolated cells
o Na,P
1
before, during and after raising bath [K ] to 20 mM. All recordings were made with pipettes filled with KF-based solution, with the fluorescent dyes fluo-3 and fura red added. During the entire period, the cells were illuminated at 10- or 20-s intervals by 1-s pulses of laser light. The initial control period varied
1
from 5 to 14 min, exposure to 20 mM [K ] from 6 to 14 min.
o
interstitial fluid of the slice and the bath in an interface 2509 pA and after washing for 15–20 min with normal
chamber is usually completed in about 30 min [6,10]. Even solution it decreased to 2344 pA Fig. 5.
1 1
though tissue [K ] probably did not reach the bath level, At the end of high [K ] treatment 1 mM tetrodotoxin
o
persistent inward currents were enhanced in all trials Figs. TTX was added to the bath in four trials. Persistent
5 and 6. The maximal amplitude and the reversal potential inward currents were substantially reduced by TTX, even
of the current shifted to a more depolarized voltage range. though the loss of the seal between pipette and cell
1
When bath [K ] was raised, the maximal persistent current membrane 10–15 min after the start of TTX administration
increased from 2325643 pA mean6S.E.M.; n 516 to precluded recording the full effect of the drug. Fig. 6A
2445658 pA at 230 mV pipette voltage, while at 220 shows average I –V curves from all four TTX-treated cells.
mV it increased from 2262654 pA to 2577661 pA. The The depression by TTX affected the persistent inward
difference was significant at both voltages P ,0.02 and currents over the entire voltage range. The curve of Fig.
P ,0.0001, respectively, by two-tailed paired t-test. An 6B was constructed by subtracting the composite I –V curve
1
even more striking change occurred at a pipette potential obtained in the presence of TTX and high [K ] from that
1
of 0 mV, at which level in normal solution the current flow taken in high [K ] without TTX, and it represents the
1
was outward, 13596174 pA, but in 10 mM [K ] it turned TTX-sensitive part of the persistent inward current. Ex-
inward, to 23356117 pA P ,0.0001. trapolation indicates that the TTX-sensitive current re-
1
With bath [K ] elevated, ‘spontaneous’ fluctuations of verses close to 142 mV, similarly to the TTX-sensitive
the holding current were noticed in several of the cells. I
of isolated neurons [37,38].
Na,P
Strongly depolarized potentials evoked irregular current fluctuations even in normal solution, which could be
described as voltage-dependent noise. The depolarization-