Department of Physiology and Pharmacology, Oregon Health Sciences
University, Portland, Oregon 97201
 |
INTRODUCTION |
The measurements of passive membrane properties,
input resistance (Rn), and membrane
time constant have been complicated by a large somatic shunt attributed
in part to impalement by the sharp electrode (Rall
1993
). This shunt can result in significant underestimates of
the passive membrane properties (Durand 1984
). Although
the principal source of this shunt remains controversial (Campbell and Rose 1997
), it has been suggested that
whole-cell recording might circumvent the impalement-induced
conductances (Blanton et al. 1989
; Edwards et al.
1989
). However, with whole-cell configuration there is a
different set of problems associated with the dialysis of the
intracellular compartment. One report in dentate granule cells
(Staley et al. 1992
) reported that there were no
time-dependent changes in either Rn or
m associated with the whole-cell recording
configuration whereas other studies presume a significant washout
effect (Spruston and Johnston 1992
). Although whole-cell
configuration is used extensively for studies of synaptic currents and
passive properties, little information is available regarding the
magnitude and time course of the washout for mammalian central neurons.
This study provides the first evidence for a widely varying time course
and magnitude of the washout effect in mammalian motoneurons. These
changes in Rn will complicate the
interpretation of synaptic currents measured at varying times after
establishing whole-cell configuration (e.g., Singer and Berger
1999
).
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METHODS |
Transverse slices were prepared from brainstems obtained from
rats that were between postnatal day 11 and 15 in age. Hypoglossal motoneurons were retrogradely labeled by injecting rhodamine dextran, under ether-induced anesthesia, into the genioglossus muscle of the
tongue two days before the experiment. On the day of the experiment, animals were initially induced with ether and deeply anesthetized with
1-2.5% isoflurane. Animals were transcardially perfused with a cold
sucrose solution containing (in mM) 215 sucrose, 0.5 CaCl2, 2 KCl, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, and 4 Mg2SO4 (pH = 7.39, 313 mOsm, bubbled with
97.5%O2-2.5%CO2). After 1 min of perfusion the rat was decapitated rapidly and its brainstem was removed and sliced into 300-µm coronal slices using a sapphire blade
(DDK) mounted on a Leica VT1000S vibratome. The slicing chamber was
cooled to ~1°C and contained the sucrose solution described above.
Slices were transferred to a holding chamber containing a low-calcium
artificial cerebrospinal fluid (ACSF) composed of (in mM) 126 NaCl, 2 KCl, 1.25 NaH2PO4, 26 NaHCO3, 1 MgCl2, 0.5 CaCl2, and 10 glucose (pH = 7.35, 300 mOsm,
30°C). After 40 min the slices were transferred to a second holding
chamber that contained a normal ACSF (2 mM CaCl2)
at room temperature.
In the recording chamber, slices were continuously perfused with normal
ACSF at room temperature (21 ± 1°C). Cells were visualized using either epifluorescence illumination or differential interference contrast (DIC) infrared video microscopy (Stuart et al.
1993
). Conventional whole-cell patch-clamp techniques
(Hamill et al. 1981
) were used in current-clamp mode to
assess the input resistance of hypoglossal motoneurons. Patch pipettes
with a tip resistance between 5 and 12 M
were filled with a solution
containing (in mM) 140 KMeSO4, 10 KCl, 5 NaCl, 2 MgCl2, 1 CaCl2, 10 HEPES,
10 BAPTA, and 2 ATP (pH = 7.35, 300 mOsm).
KMeSO4 was chosen instead of the more commonly
used K-gluconate because of reports that the former preserves neuronal
excitability much more effectively than the latter (Velumian et
al. 1997
; Zhang et al. 1994
). Current-clamp recordings were made with an Axopatch 1-D patch-clamp amplifier. The
data were low-pass filtered at rates between 1 and 2 kHz and digitized
at rates between 2.5 and 5 kHz.
Immediately after the whole-cell configuration was attained, the
resting membrane potential was recorded. The value of the resting
potential was monitored regularly throughout the session. All
statistics are expressed as the mean ± SD and significance was
assessed using the Student's t-test.
 |
RESULTS |
This study is based on 24 developing hypoglossal motoneurons.
These neurons had stable resting membrane potentials for at least 15 min and elicited a sustained discharge of action potentials in response
to maintained current depolarization. The mean resting potential of
these neurons was
64.4 ± 4.7 mV and the mean maximum firing
frequency evoked by maintained depolarization was 37.6 ± 14.8 Hz.
If a neuron was unable to elicit such sustained firing and/or its
resting membrane potential became >5 mV more depolarized from its
initial value, the recording was terminated.
Figure 1 shows typical recordings
obtained from a hypoglossal motoneuron 0, 15, and 33 min after the
whole-cell configuration had been obtained. The response of the neuron
at the three times to a 300 pA maintained depolarization (1 s duration)
is shown at the top of each panel. At each time the neuron responded
with a sustained discharge of action potentials at a frequency of ~40 Hz. The bottom panel illustrates responses to a series of
hyperpolarizing current steps recorded immediately after the traces
above. Each hyperpolarizing step (390 ms duration) was made from the
resting membrane potential and its magnitude increased sequentially
from 0 to
300 pA in steps of
50 pA. The "sag," evident in the
response to the larger hyperpolarizations, reflects activation of an
inwardly rectifying cation channel and was observed in 23 of 24 neurons analyzed. The magnitude of the sag, determined by subtracting the peak
potential from the membrane potential at 385 ms for the maximum step
used, was 2.3 ± 2.6 mV, which represents only a 10% decrease in
resistance across the cells sampled.

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Fig. 1.
Whole-cell current-clamp recordings obtained from a 15-day-old
hypoglossal motoneuron. Top: response of neuron to a 1-s
maintained depolarizing current (300 pA amplitude) recorded 0, 15, and
33 min after the whole-cell configuration was attained.
Bottom: average of 3 series of hyperpolarizing current
steps made immediately after depolarizing step above. Calculated input
resistance for the cell at each time is listed at bottom of each
panel.
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Rn was determined from the slope of a
linear fit of the relationship between the peak change in membrane
potential (
Vm) and the magnitude of
the injected current. The top panel of Fig.
2 shows such relationships for the
recordings illustrated in Fig. 1. The bottom panel of Fig. 2 shows how
the Rn of the neuron illustrated in
Fig. 1 changes with time after the whole-cell configuration had been
obtained. Initially, the Rn was 73 M
and increased gradually, reached a maximum of 101 M
at 33 min,
and remained at this value for ~5 min before it decreased rapidly.
This decrease can be attributed to seal breakdown and rapidly resulted
in the cell losing its ability to generate action potentials (not
shown).

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Fig. 2.
Time-dependent changes in input resistance
(Rn) after attainment of whole-cell
configuration. Top: plots membrane potential as a
function of injected current to derive slope,
Rn. Bottom: calculated
resistance is plotted as a function of time elapsed from attainment of
whole-cell configuration.
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A time dependent increase in Rn was
observed in all 24 of the neurons studied. The mean initial
Rn was 119.5 ± 71.3 M
and increased in a time-dependent manner to a mean maximum value of 162.8 ± 103.8 M
. The paired Student's t-test
revealed that this increase was highly significant (P < 0.0001).
The time course over which the increase in
Rn occurred varied greatly between
cells. Figure 3A shows the
time-dependent increase in input resistance, normalized to the maximum
value attained, plotted for two further neurons. As indicated by these
representative cells, a number of neurons reached their maximum input
resistance within 10 min whereas others took much longer. There was no
relationship between the size of the change in
Rn and the rate at which this change
occurred. Table 1 summarizes the
responses of the 24 cells recorded based on the time it took for the
maximum Rn to be attained. Although
some neurons reached a maximum in <10 min, it should be noted that the
recordings lasted much longer and were terminated only when the resting
membrane potential changed by more than +5 mV or the cell no longer
generated sustained discharge. Table 1 demonstrates that the rate of
increase in input resistance was unrelated to either the resting
membrane potential or maximum firing frequency observed in each group
of neurons.

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Fig. 3.
Variation in time course of increase in Rn
and results obtained with perforated-patch recordings.
A: 2 representative neurons selected to illustrate
different time courses (0-10 min, >21 min) over which increase
occurred. Rn has been normalized to a
maximum equal to 1.0. B: data obtained while using the
perforated-patch variation of patch-clamp technique.
Top: spiking patterns obtained to a depolarizing current
injection at t = 0 and 13 min after whole-cell
configuration was obtained. Middle: responses to
hyperpolarizing currents at t = 0 and 13 min.
Bottom: plots Rn, normalized
to its maximum value, against time. In 4 neurons recorded using this
technique, no increase in Rn was ever
observed.
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 |
DISCUSSION |
The introduction of patch-clamp recording has substantially
improved our ability to resolve small synaptic currents (Edwards 1995
; Edwards et al. 1989
) in the neurons of the
mammalian CNS. One consequence of creating a continuity between the
cytoplasm and the electrode solution is the movement of substances from the smaller volume of the cytoplasm into the larger volume of the
electrode that are important for maintaining the resting membrane state. G-protein-regulated conductances in hypoglossal motoneurons are
under the control of various neurotransmitters (Bayliss et al.
1997
) and these currents would be altered by the dialysis. The
values of membrane properties like Rn
and membrane time constant in the hippocampus have been found to be
greater using either whole-cell (Staley et al. 1992
) or
perforated-patch (Spruston et al. 1994
) than those found
using the sharp electrode. The results described above agree with such
findings. In identical preparations, Rn determined using sharp electrode
recording was 47 ± 19 M
(Núñez-Abades et
al. 1993
) as compared with 120 ± 71 M
that is reported
here. Such an increase is in line with the approximate two- to fourfold increase found in hippocampal CA1 and CA3 neurons (Spruston et al. 1994
).
In contrast to our study, significant time-dependent changes in
Rn were not observed in dentate
granule cells of the rat hippocampus (Staley et al.
1992
). However, closer inspection of their Fig. 6 reveals
evidence for such an increase in their data. No time-dependent changes
in Rn were reported in a recent study
using whole-cell recording in rat spinal ventral horn neurons
(Thurbon et al. 1998
). On the basis of the 4 of 10 neurons studied, these authors concluded "the results resolve the
issue of a somatic shunt conductance for motoneurons, relegating it to
a microelectrode impalement artifact." Without electrode damage,
there would be no somatic shunt and the motoneuron could be modeled
using uniform resistivity. However, the majority of the cells from this
study did require a somatic shunt to fit their model and these data are
more consistent with a role for potassium conductances in generating
the somatic shunt as proposed for cat cervical motoneurons studied in
vivo (Campbell and Rose 1997
). It is some of these
conductances modulated by second messenger systems that could be the
principal target of the washout phenomenon.
These results indicate that conventional whole-cell recording is not
the best choice for experiments where
Rn is being measured. The rise in
resistance is variable in both its absolute magnitude and its time
course. One explanation for this increase in
Rn is that the membrane is slowly
resealing under the patch electrode. In this scenario, the magnitude of
the instantaneous change in membrane potential would become much larger
as the contribution of the RC time-constant associated with the
electrode begins to overwhelm the time-constant associated with the
membrane. As clearly indicated by Fig. 1, this was not found to be the
case. Moreover, a preliminary report (Robinson and Cameron
1998
) suggests that such increases in input resistance can
apparently be avoided by using perforated-patch. In such recordings no
rise in input resistance was ever observed (n = 4).
Conversely, it remained at a constant value (n = 1) or
more typically (n = 3; Fig. 3B) decreased
slowly over time, presumably a result of the incorporation of more
Nystatin pores into the membrane patch.
There are increasing numbers of reports that examine membrane
properties and/or synaptic currents using conventional whole-cell recording and it should be cautioned that these results might be very
difficult to interpret if the neuronal populations studied exhibit a
similar response to cellular dialysis.
This research was supported by National Institute of Child Health
and Human Development Grant HD-22703 and by SIDS Foundation Megan's
Run, Wilsonville, OR.
Address for reprint requests: D. W. Robinson, Physiology and
Pharmacology, L334, Oregon Health Sciences University, 3181 S.W. Sam
Jackson Park Rd., Portland, OR 97201.
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
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1734 solely to indicate this fact.