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The Journal of Neuroscience, July 1, 2000, 20(13):4855-4863
Excitatory Role of the Hyperpolarization-Activated Inward Current
in Phasic and Tonic Firing of Rat Supraoptic Neurons
Masoud
Ghamari-Langroudi and
Charles
W.
Bourque
Centre for Research in Neuroscience, Montreal General Hospital and
McGill University, Montreal, Quebec H3G 1A4, Canada
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ABSTRACT |
The properties and functional roles of the
hyperpolarization-activated inward current
(IH) in magnocellular neurosecretory cells (MNCs) were investigated during sharp microelectrode recordings from supraoptic neurons in superfused explants of rat hypothalamus. Under current clamp, voltage responses to hyperpolarizing current pulses featured depolarizing sags that were abolished by the
IH blocker ZD 7288. Under voltage clamp,
subtraction of current responses to hyperpolarizing steps recorded in
the absence and presence of ZD 7288 was used to investigate the
properties of IH. Current-voltage analysis
revealed that steady-state IH amplitude
increases with hyperpolarization, with half-maximal activation of the
underlying conductance occurring at 
78 mV. The time course of
activation of IH during hyperpolarizing
steps was monoexponential with time constants (100-800 msec)
decreasing with hyperpolarization. The effects of ZD 7288 on
IH were slow (
, ~15 min), irreversible, and half-maximal at 1.8 µM. When tested on continuously
active MNCs, application of 30-60 µM ZD 7288 caused a
significant reduction in firing rate. In phasically active MNCs, the
drug decreased burst duration and intraburst firing frequency and
caused an increase in the duration of interburst intervals. These
effects were accompanied with a small hyperpolarization of the membrane
potential. In contrast, ZD 7288 had no effect on spike duration, on the
amplitude of calcium-dependent afterpotentials, or on the frequencies
and amplitudes of spontaneous synaptic potentials. These results
confirm the presence of IH in MNCs of the
rat supraoptic nucleus and suggest that the presence of this
conductance provides an excitatory drive that contributes to phasic and
tonic firing.
Key words:
vasopressin; oxytocin; neurohypophysis; neurosecretory
cell; burst firing; hypothalamus; ZD 7288
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INTRODUCTION |
Hypothalamic magnocellular
neurosecretory cells (MNCs) are responsible for the release of either
vasopressin (VP) or oxytocin (OT) into the blood (Poulain and Wakerley,
1982
). After their synthesis in MNC somata, peptides are packaged in
vesicles and transported to axon terminals in the neurohypophysis
(Brownstein et al., 1980) where exocytosis is triggered by the arrival
of action potentials (Dreifuss et al., 1971). In the rat, both OT- and
VP-releasing MNCs initially respond to hyperosmolality (Brimble and
Dyball, 1977; Wakerley et al., 1978) by increasing their firing rate.
During sustained or strong stimulation, however, an increasing proportion of VP-MNCs adopt phasic firing, a pattern comprising alternating periods of activity (7-15 Hz) and silence lasting tens of
seconds each. Studies using the neurohypophysis in vitro have shown that increases in firing frequency potentiate the amount of
peptide secreted per action potential (Dreifuss et al., 1971). Over the
same range of frequencies, however, VP release is maximized by
stimulation patterns mimicking phasic firing (Dutton and Dyball, 1979;
Bicknell and Leng, 1981), presumably because of the reversal of
secretory fatigue during periods of quiescence (Bicknell et al., 1984).
The firing pattern of MNCs, therefore, is precisely matched to the
overall demand for hormone release (Bicknell, 1988).
Studies in many types of neurons have shown that one of the key
conductances controlling rhythmic bursting is the slow
hyperpolarization-activated inward current
(IH; Pape, 1996). Although a previous
study has shown that guinea pig MNCs express
IH (Erickson et al., 1993), the
presence of this current in rat MNCs is controversial. Classically, IH is blocked by low millimolar
concentrations of extracellular Cs+
(Halliwell and Adams, 1982). At voltages where
IH is active, therefore, bath
application of Cs+ normally causes the
appearance of an outward current. Surprisingly, application of
Cs+ to rat MNCs resting 5-25 mV below
action potential threshold causes depolarization (Stern and Armstrong,
1997; Ghamari-Langroudi and Bourque, 1998). Additionally, the presence
of IH is classically associated with
the generation of depolarizing sags of increasing amplitude when
hyperpolarizing current pulses of increasing magnitude are applied from
voltages near rest (McCormick and Pape, 1990). In rat MNCs, however,
depolarizing sags associated with voltage responses to negative current
pulses applied from 50 mV become smaller with hyperpolarization
(Stern and Armstrong, 1995, 1997). These observations suggest that rat
MNCs lack IH or that these cells
express superimposed membrane currents that complicate the detection of
IH under experimental conditions.
The bradycardic agent ZD 7288 was recently shown to specifically block
IH in neurons of the substantia nigra
(Harris and Constanti, 1995). The presence or absence of
IH in rat MNCs, therefore, could be
determined by examining the effects of this compound on depolarizing sags evoked by hyperpolarizing current steps. Moreover, if
IH is present, digital subtraction of
current traces recorded in the absence and presence of ZD 7288 could
provide information concerning its kinetics and voltage dependency. In
this study, we reveal that rat MNCs indeed express
IH and that the current plays an
important role in the control of electrical activity.
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MATERIALS AND METHODS |
Preparation of superfused explants. Hypothalamic
explants were prepared as described previously (Bourque, 1988).
Briefly, male Long-Evans rats (150-300 gm) were restrained (5-10
sec) in a soft plastic cone and killed by decapitation using a small
rodent guillotine (model 51330; Stoelting Company, Wood Dale, IL). This tissue-harvesting protocol has been approved by the McGill University Animal Care Committee. The brain was then rapidly removed from the
cranial vault. A block of tissue (~8 × 8 × 2 mm)
comprising the basal hypothalamus was excised using razor blades and
pinned, ventral side up, to the Sylgard base of a
temperature-controlled (33-35°C) superfusion chamber. Within 2-3
min of decapitation, explants were being superfused (0.5-1 ml/min)
with an oxygenated (95% O2 and
5% CO2) artificial
CSF (ACSF; see below) delivered via a Tygon tube placed over the
caudal portion of the optic tract. The arachnoid membrane covering the
ventral surface of the supraoptic nucleus was removed using fine
forceps, and a cotton wick was placed at the rostral tip of the explant
to facilitate drainage of ACSF.
Solutions and drugs. The ACSF (pH 7.4; 295 ± 3 mOsmol/kg) was comprised of (mM): NaCl, 121;
MgCl2, 1.3; KCl, 3; NaHCO3,
26; glucose, 10; CaCl2 2.5 (all from Fisher
Scientific, Pittsburgh, PA). The ACSF was supplemented, where indicated
in the text, with 0.3-0.6 µM tetrodotoxin (TTX; Sigma,
St. Louis MO) and/or tetraethylammonium Cl (TEA; 1-3 mM;
Sigma). The IH blocker ZD 7288 (from
Tocris Cookson, Ballwin, MO) was prepared as a 30 mM stock solution (in H2O)
and stored at 4°C. The effects of the drug were examined by bath
application of ACSF containing a dilution of the stock solution.
Electrophysiology. Intracellular recordings were obtained
using sharp micropipettes prepared from glass capillary tubes (1.2 mm
o.d.; A.M. Systems, Everett, WA) pulled on a P87 Flaming-Brown puller
(Sutter Instruments, Novato, CA). Pipettes were filled with 2 M potassium acetate, yielding a DC resistance of 70-150 M
relative to a Ag-AgCl wire electrode immersed in ACSF. Recordings of membrane voltage (dc-5 kHz) and current (dc-0.3 kHz) were obtained through an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA).
Voltage recordings were performed in continuous current clamp ("bridge") mode, whereas current recordings were performed using the discontinuous single-electrode voltage-clamp (dSEVC) mode. Switching frequencies in dSEVC mode were adjusted (2-3.5 kHz) to
assure that a complete decay of the electrode potential was achieved
between periods of current injection. Because no current is injected
during the voltage-sampling period, there are no problems with series
resistance when using dSEVC. To demonstrate clamp performance, all
figures showing voltage-clamp data incorporate the actual voltage
traces sampled during the experiment, rather than a copy of the
template protocol generated by the computer. Signals acquired during
each experiment were displayed on a chart recorder and digitized (44 kHz; Neurodata, Delaware Water Gap, PA) for storage onto videotape.
Current and voltage pulses were delivered through an external pulse
generator, or via a Labmaster interface driven by Clampex 5.51 (Axon
Instruments) running on an AT-compatible computer. Current
traces were digitized at a rate of 0.67 kHz. Averaging of current
traces, and digital subtraction, were performed using Clampfit 6.0 (Axon Instruments).
Measurement of membrane potential in active cells. The
resting membrane potential observed during spontaneous firing was
defined as the mean stationary voltage observed during interspike
intervals, excluding the decaying phase of hyperpolarizing
afterpotentials and the depolarizing ramps that precede the discharge
of each action potential. However, the detection of stationary phases becomes difficult during periods of relatively fast firing (5-15 Hz).
We therefore used the following method to determine the mean resting
potential of active cells in an unbiased manner. All-points histograms
(bin width 0.04-0.1 mV) of voltage excerpts digitized at 4-5 kHz
(4-10 sec long) were constructed using Fetchan 6.0 (Axon Instruments).
The frequency distribution of the voltage samples obtained in this
manner showed two clear peaks. The most negative peak registered the
brief stationary phase at the peak of the hyperpolarizing
afterpotentials whereas a larger, slightly more depolarized, peak
reflected the stationary phases interspersed in the recording (see Fig.
9). Resting membrane potential was thus defined as the voltage
corresponding to this peak.
Synaptic potential analysis. Continuous voltage segments
(DC, 5 kHz) lasting 8-12 min were digitized at 5 kHz using Fetchex 5.51 (Axon Instruments). Data files were imported into Axograph 4.0 (Axon Instruments) running on a MacIntosh computer. Spontaneous EPSPs (sEPSPs) and IPSPs (sIPSPs) were detected using a
cell-specific event template and a variable signal-to-noise threshold
criterion. The amplitude of all events (relative to baseline) was
measured automatically. Cumulative probability distributions of
synaptic potential amplitude were constructed using Kaleidagraph 3.08 (Synergy Software). Differences between amplitude distributions were
sought by comparing absolute amplitudes at 50% probability (i.e., the median amplitude). The groups of events recorded under different conditions comprised between 54 and 2849 events (mean, 574 ± 143).
Estimation of GH. The voltage
dependency of GH can be assessed
experimentally by plotting the relative amplitude of current tails
evoked at a fixed potential after pulses delivered to different conditioning potentials (McCormick and Pape, 1990). In MNCs, however, relatively large currents are activated or deactivated after
termination of voltage steps to negative potentials. In particular, the
amplitude of the transient K+ current
measured at 50 mV after a prepulse to 120 mV can exceed 1.5 nA
(Bourque, 1988), a value ~100 times greater than that of the
IH tail expected to result from the
same protocol. We therefore simply estimated
GH as the amplitude of
IH measured at various potentials
(V) divided by the driving force (V EH), where
EH is the reversal potential of
IH. Moreover, because we could not measure EH directly, the value was
arbitrarily set at 35 mV, a value reflecting the median
EH reported during sharp electrode voltage-clamp studies in a variety of cell types (Pape, 1996).
Dose-response analysis of the effects of ZD 7288. The time
course of blockade of IH by ZD 7288 was slow and monoexponential (mean , ~15 min; see Results).
Therefore, only cells in which the effects of a maintained dose of ZD
7288 could be monitored for >15 min were included in the
dose-response analysis. The amplitude of
IH measured in cells from which data
were obtained over a period exceeding 60 min in ZD 7288 (i.e., 4 times
) were accepted as is. In trials in which the application of ZD 7288 lasted between 15 and 60 min, the amplitude of
IH that would have been blocked at
equilibrium (IH
) was estimated as
IH = IH(t)/(1 e
t/15), where
IH(t) is the amplitude of
IH measured t min after the onset of the application.
Statistics. Throughout the paper, averaged data are
expressed as mean ± SEM. Differences between mean values recorded
under control and test conditions were evaluated using a paired
t test, if indicated, and were considered significant when
p < 0.05.
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RESULTS |
The data presented below were obtained during intracellular
recordings made from 68 supraoptic nucleus neurons impaled with sharp
microelectrodes in superfused explants of rat hypothalamus. These cells
had resting membrane potentials more negative than 50 mV, input
resistances exceeding 180 M, and fired action potentials with
amplitude exceeding 60 mV when measured from baseline. Each of these
cells also displayed frequency-dependent spike broadening (Andrew and
Dudek, 1985; Bourque and Renaud, 1985b) and transient outward
rectification (Bourque, 1988) when examined from initial membrane
potentials less than 75 mV. These combined characteristics have been
shown to be specific to magnocellular neurosecretory neurons, but not
to neighboring non-neuroendocrine cells, during intracellular
recordings in vitro (Renaud and Bourque, 1991; Tasker and
Dudek, 1991) and in vivo (Bourque and Renaud, 1991; Dyball et al., 1991).
Effects of ZD 7288 in current clamp
Previous studies have shown that deactivation of the
K+ conductance responsible for sustained
outward rectification in MNCs can contribute to the production of sags
during voltage responses to hyperpolarizing steps applied from holding
membrane potentials near 50 mV (Stern and Armstrong, 1995, 1997). To
minimize the steady-state contribution of this conductance, therefore,
we examined the effects of current injection in cells held at voltages
near or below 65 mV. Under these conditions, application of prolonged (1-4 sec) negative current pulses (100 to 200 pA) caused
hyperpolarizing electrotonic voltage responses consistently
superimposed by depolarizing sags in each of 12 cells tested. Moreover,
as illustrated in Figure 1A, bath application of
ZD 7288 (30-60 µM; n = 10)
caused a slow (, ~13 min) and progressive reduction of the
amplitude of the sag (Fig. 1B). These results suggest
that supraoptic MNCs in rats express the hyperpolarization activated
current IH.
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Figure 1.
Effects of ZD 7288 in current clamp.
A, Intracellular recording (initial
Vm, 63 mV) from a supraoptic nucleus MNC
showing voltage responses (bottom) to current pulses
(top) before and after the application of ZD 7288 (bar). The traces are time-expanded at the beginning and
end of the record to highlight the presence of a depolarizing sag in
the voltage response under control conditions (left) and
its gradual disappearance (middle) and eventual reversal
(right) in the presence of ZD 7288. The amplitude of the
sag evoked by each pulse was measured by comparing the voltage at the
end of the pulse with that recorded 60 msec after the onset of the
pulse (dashed lines). The gap indicates the presence of
a 34 min interval. B, Time course of the effect of ZD
7288 on the sags recorded from the cell shown in A. The
dashed line is a monoexponential fit though the data
points ( = 12.6 min; r = 0.98).
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Isolation of IH in MNCs
Because IH is superimposed by
other time- and voltage-dependent currents expressed at subthreshold
voltages, the properties of the underlying conductance
(GH) could not be extracted directly from a simple analysis of voltage traces recorded under current clamp
(as above) or of current traces recorded under voltage clamp (Fig.
2A). The properties of
IH in MNCs, therefore, were analyzed using a subtraction procedure in which averaged (n 
2) current responses to voltage steps applied under control conditions
were digitally subtracted from traces recorded in the presence of ZD 7288 (Fig. 2B). As shown in Figure 2C,
subtraction analysis revealed that bath application of ZD 7288 progressively blocked a slow-activating, noninactivating inward current
evoked by hyperpolarizing voltage steps. The time course of block of
IH by ZD 7288 was monoexponential (Fig. 2D). Similar to its effect on depolarizing sags
recorded under current clamp (Fig. 1B), the mean time
constant characterizing the time course of ZD 7288-evoked block of
IH was 14.8 ± 1.4 min (n = 5). In three cells in which the amplitude of the
IH blocked by ZD 7288 was monitored
during washout, the amplitude of the blocked current 60 min after the
onset of wash (44.3 ± 5 pA) represented 96% of the maximum
IH blocked in the presence of the drug
(47 ± 5 pA).
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Figure 2.
Isolation of IH by
digital subtraction of the ZD-sensitive current in MNCs.
A shows the average (n = 5 trials
per trace) current responses (bottom) to 80 mV
hyperpolarizing voltage commands (top) applied from a
holding potential of 40 mV. The traces shown were recorded before
(control) and ~40 min after adding 30 µM ZD 7288 to the
bath. The experiment was performed in the presence of 0.5 µM TTX and 3 mM TEA to block voltage-gated
Na+ and K+ currents,
respectively. B shows the difference current
(IH) obtained by subtracting the
current traces shown in A. The traces in
C are ZD 7288-sensitive current traces
(bottom) recorded in a different cell at a variety of
intervals after beginning the application of ZD 7288. The graph in
D plots the amplitudes of IH
measured by digital subtraction, as a function of time in ZD 7288, for
five cells in which the time course of block was tracked over a period
>60 min. Different symbols refer to different cells. The solid
line is a monoexponential fit through the data ( = 14.8 min; r = 0.96).
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Voltage dependency of IH
The voltage dependency of IH was
examined by measuring the amplitude of the ZD 7288-sensitive current
evoked by hyperpolarizing voltage steps applied from a holding
potential near rest. Typically, individual current-voltage
(I-V) relations were obtained by
delivering a series of prolonged (2-3 sec) voltage steps to values
between 115 and 40 mV at sufficiently slow frequency (
0.05 Hz) to
allow complete interpulse deactivation of
IH. Such trials were repeated at
regular intervals before and during the application of ZD-7288 (Fig.
3A). Digital subtraction of
averaged current traces obtained in control from those recorded in the
presence of ZD 7288 (Fig. 3B) yielded a family of current
traces reflecting the activation time course and amplitude of the
IH recorded at each voltage (Fig. 3C). The steady-state amplitude of
IH was measured at each of the
voltages. These data were then averaged across the population of cells
tested (n = 11), and the mean value of
IH (± SEM) was plotted as a function
of voltage (Fig. 4A).
The resultant current-voltage (I-V)
relation indicates that IH is a
voltage-dependent inward current with an apparent activation threshold
of ~60 mV. For each cell the conductance
(GH)-voltage relation of
IH was estimated from the value of
IH assuming a reversal potential
(EH) of 35 mV (see Materials and
Methods) and GH = IH/(V EH), where V is the voltage
during the test pulse. The mean
GH-V relation observed in
the group of MNCs tested (n = 11) is shown in Figure
4B. The data were well fitted (r = 0.994; n = 11) using the equation
GH(V) = 1/(1 + e(V V1/2)/b), where
GH(V) is the fraction of maximal
GH observed at V,
b is the slope factor, and V1/2
is the half-maximal voltage. The mean
GH V relation reveals a
V1/2 of 78 mV and a slope factor of 12 (Fig. 4B).
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Figure 3.
Current-voltage (I-V)
analysis of IH in MNCs.
A, Chart recording showing current responses of an MNC
to consecutive I-V trials consisting of
a series of 2.5 sec hyperpolarizing voltage steps to potentials between
114 and 49 mV (Vh = 40 mV). After
14 min of control recording ZD 7288 was bath-applied
(bar). B shows at high gain, averaged
(n = 4 trials per set) current responses
(bottom) to voltage steps (top) recorded
before (control) and after the application of ZD 7288 in the same cell.
Note the presence of slow current relaxations in the traces recorded in
the presence of ZD 7288. The reversal in polarity of the latter events
at approximately 87 mV suggests that they reflect the deactivation of
a superimposed voltage-dependent K+ current. The
traces in C are the digitally subtracted ZD
7288-sensitive currents evoked by the steps to each voltage
(indicated).
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Figure 4.
Voltage-dependent activation of
IH in MNCs. A, plots the mean
(± SEM) steady-state amplitude of IH
recorded at different voltages in 11 MNCs. Values at 5 mV increments
between 40 and 120 mV were derived by interpolation of the
I-V curves generated in each cell using
a protocol such as that shown in Figure 3. B, The
current (IH) data shown in
A were converted into conductance
(GH) using the equation
GH = IH/(V + 35) where
V is the test voltage. The solid line is
the best fit through the data points using the Boltzmann equation
(V1/2 = 78 mV;
k = 12; see "Voltage dependency of
IH" for details).
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The kinetics of activation of IH
The time course of activation of
IH was obtained from an analysis of
the rising phase of the ZD 7288-sensitive current evoked by
hyperpolarizing steps to various voltages. As shown in Figure 5A, ZD 7288-sensitive current
traces were well fitted by a single exponential function of the form
At = A(1 et/
), where
At is the amplitude of
IH at time t,
A the amplitude of
IH at steady state, and is the
activation time constant. Figure 5B reveals that mean values
of decreased from ~800 msec at 60 mV to ~100 msec at 120 mV
(n = 11).
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Figure 5.
Voltage dependency of
IH kinetics in MNCs.
A shows IH traces
(bottom) evoked by steps to various voltages
(V; protocol shown above and as indicated above each
current trace). Each current trace is superimposed by a solid
line extending to the right showing the best monoexponential
fit of the data. The time constants used in the fits () are
indicated beside each trace. B, Plot of the mean (±SEM)
IH activation time constants measured in 11 MNCs. Values at 5 mV increments between 60 and 120 mV were derived
by interpolation of individual -V curves generated for each cell.
The solid line was fitted by eye.
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Dose dependency of IH block by
ZD 7288
The dose dependency of inhibition of
IH by ZD 7288 was quantified by
measuring the absolute steady-state amplitude of
IH at 105 mV
(IH), which was blocked by
different concentrations of ZD 7288 (see Materials and Methods). The
mean amplitudes of IH were then
plotted as a function of the logarithm of the concentration of ZD 7288 (n = 27; Fig. 6). The
data were fitted by the equation
IH(C) = IH(MAX) × (1 [1/(1 + [C/C0.5]b)]),
where C is the concentration of ZD 7288, IH(MAX) is the maximal IH available at 105 mV,
IH(C) is the
value of IH at a given
C, and C0.5 is the
concentration of ZD 7288 resulting in half-maximal block. The best fit
through the data (r = 0.983) revealed values of
IH(MAX) of 72 pA and
C0.5 of 1.8 µM.
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Figure 6.
Dose dependency of the block of
IH in MNCs by ZD 7288. The
graph plots the mean (± SEM) absolute steady-state
IH amplitude measured by digital subtraction
of current traces evoked by voltage steps to 110 mV commanded in the
absence and presence of different concentrations of ZD 7288. The
solid line shows the best fit through the data points
(IC50 = 1.84 µM; r = 0.98). The number of observations made at each concentration is shown
in parentheses.
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Contribution of IH at subthreshold
membrane potentials
Under current-clamp conditions, bath application of 30-60
µM ZD 7288 to silent MNCs held at initial membrane
potentials between 66 mV and 111 mV provoked a robust membrane
hyperpolarization in each of four cells tested (Table
1). However when applied to six other
cells held below spike threshold (approximately 52 mV), but above
65 mV, ZD 7288 only had a small, statistically insignificant,
hyperpolarizing effect (Fig. 1A, Table 1).
Role of IH in spontaneously
active neurons
The possible role of IH in the
regulation of firing in MNCs was evaluated by examining the effects of
ZD 7288 on 11 spontaneously active cells. In continuously firing cells
(n = 6), bath application of 30-60
µM ZD 7288 consistently reduced the mean rate
of action potential discharge (Fig. 7).
When measured 15-30 min after the onset of the application, the mean
firing rate of the cells had decreased to 4.3% of control (Table 1).
Moreover, in the presence of the drug, the mean resting potential of
the cells (see Materials and Methods) was significantly hyperpolarized
compared to control (Table 1). Bath application of 30-60
µM ZD 7288 also had profound effects on
spontaneously phasically firing MNCs (n = 5; Fig.
8). When averaged between 15 and 30 min
after the onset of the application, the presence of ZD 7288 was found
to cause a 41% decrease in the duration of bursts, a 77% decrease in
the steady-state intraburst firing frequency, and a 19% increase in
the duration of interburst silent intervals (Table 1). Whereas the mean
membrane potential observed during the steady-state portion of the
plateau phase of each burst was significantly hyperpolarized in the
presence of ZD 7288 (Fig. 9), the minimum
voltage achieved between bursts was not affected (Table 1).
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Figure 7.
Effects of ZD 7288 on continuously
active MNCs. A shows a voltage recording obtained from a
continuously active MNC. Application of ZD 7288 (bar)
resulted in a progressive decrease in firing rate. B,
Rate meter plot of the action potentials recorded from the cell shown
in A (bin width, 1 sec).
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Figure 8.
Effects of ZD 7288 on phasically active MNCs. The
figure shows a recording made from a phasically active MNC. The record
is broken up into seven consecutive segments in which the top
trace shows membrane voltage, and the bottom
trace plots firing rate (bin width, 1 sec).
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Figure 9.
Effects of ZD 7288 on membrane potential during
phasic firing. The panels on the right show high gain
examples of the electrical activity (spikes truncated) recorded from a
single cell during the steady-state phase of phasic bursts recorded in
the absence (control) and presence of 60 µM ZD 7288. All-points histograms plotting the distribution of the voltage samples
comprising each trace are shown on the left. In addition
to causing a clear decrease in firing frequency, the histograms reveal
that ZD 7288 caused a significant hyperpolarization of the membrane
potential (see Materials and Methods for details).
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The selectivity of the effects of ZD 7288
Previous studies have suggested that ZD 7288 selectively inhibits
IH without measurable effects on a
number of other cell properties (Harris and Constanti, 1995; Williams
et al., 1997; Gasparini and DiFrancesco, 1997). However when averaged
15-30 min after the onset of the application, we found that the
presence of 30-60 µM ZD 7288 caused a small,
but significant, decrease in spike amplitude (Table
2). In contrast to its effects on
IH, however, the reduction of spike
amplitude caused by ZD 7288 was readily reversed with a wash period
lasting 20-40 min. These observations suggest that this compound may
have additional effects on voltage-gated Na+ channels,
Ca2+ channels, and/or repolarizing
K+ channels. We therefore examined if the
excitability of the cells was significantly reduced in the presence of
ZD 7288. As shown in Table 2, the mean depolarizing current pulse
(40-80 msec) amplitude required to evoke a constant (1-3) number of
action potentials from a fixed voltage between 65 mV and spike
threshold was not affected by ZD 7288. Moreover, in spontaneously
active neurons, firing resumed after compensating for the
hyperpolarizing effects of ZD 7288 with depolarizing current injection
(data not shown). Because the rate and pattern of firing in MNCs is
influenced by a number of Ca2+-dependent
conductances (Hu and Bourque, 1992; Li et al., 1995; Stern and
Armstrong, 1995, 1997; Kirkpatrick and Bourque, 1996; Li and Hatton,
1997; Ghamari-Langroudi and Bourque, 1998; Greffrath et al., 1998), we
also examined if the effects of ZD 7288 on spontaneous firing might be
attributable to indirect effects on such properties. Previous studies
have shown that changes in Ca2+ influx
provoke changes in the duration of action potentials (Bourque and
Renaud, 1985a; Kirkpatrick and Bourque, 1991), as well as in the
amplitude of the Ca2+-dependent
hyperpolarizing afterpotential (HAP; Bourque et al., 1985) and
depolarizing afterpotential (DAP; Bourque, 1986; Li et al., 1995; Li
and Hatton, 1997). Changes in these parameters may therefore reflect
changes in Ca2+ influx. As shown in Table
2, however, bath application of 30-60 µM ZD
7288 for 15-30 min had no effect on spike duration or on the amplitude
of the HAP or DAP. Finally, we examined if the effects of ZD 7288 on
spontaneous firing could be attributable to changes in afferent
synaptic drive. As shown in Table 2, however, neither the amplitudes
nor the overall frequencies of sIPSPs or sEPSPs were significantly
affected by the drug.
| |
DISCUSSION |
As explained earlier, previous observations have made it unclear
if MNCs in the rat supraoptic nucleus express the
hyperpolarization-activated inward current
IH. In the present study, we show that
ZD 7288, a selective blocker of IH,
inhibited the time-dependent inward current (voltage clamp;
n = 33) and depolarizing sags (current clamp;
n = 12) evoked by hyperpolarization, as well as
spontaneous firing (n = 11), in all of the MNCs tested.
Because it is likely that both OT and VP MNCs were sampled in our
recordings, it would appear that both types of cells indeed express
IH.
Isolation of IH in MNCs
To characterize the properties of
IH, MNCs were stepped to potentials
between 40 and 120 mV under voltage clamp (Fig. 3). However,
because other time- and voltage-sensitive conductances are known to be
active over this range (Bourque, 1988; Cobbett et al., 1989; Stern and
Armstrong, 1995, 1997; Fisher and Bourque, 1995), current relaxations
evoked during such voltage steps are not likely to reflect
IH exclusively. To isolate
IH from other "contaminating"
currents, therefore, we digitally subtracted current responses to
voltage steps evoked in the absence and presence of ZD 7288, a
selective blocker of IH (Harris and
Constanti, 1995). The necessity to use a subtraction procedure, rather
than a simple analysis of current traces recorded under control
condition, was confirmed by the presence of significant current
relaxations during responses to voltage steps recorded in the absence
of IH (Fig. 3B). Finally,
the decision to use ZD 7288 rather than
Cs+, another well known blocker of
IH (Halliwell and Adams, 1982), was
guided by the fact that the block of
IH by Cs+
is known to be voltage-dependent (Pape, 1996) and because
Cs+ affects other conductances in MNCs
(Ghamari-Langroudi and Bourque, 1998).
Blockade of IH by ZD 7288
The progression of the blockade of
IH in MNCs was monitored for periods
of up to 2 hr during bath application of ZD 7288. The block of
IH by ZD 7288 was found to be both
concentration- and time-dependent. The half-maximal blocking
concentration was 1.8 µM (Fig. 6), a value that
agrees well with the reported effects of ZD 7288 on
IH in SNC neurons (2 µM; Harris and Constanti, 1995) and CA1 cells
of the hippocampus (10.5 µM; Gasparini and
DiFrancesco, 1997).
The time course of blockade of IH by
ZD 7288 was slow and could be well fitted by a monoexponential function
with a time constant of ~15 min (Fig. 2). Slow effects of ZD 7288 on
IH have also been reported in
hippocampal (Gasparini and DiFrancesco, 1997), SNC (Harris and
Constanti, 1995), and trigeminal neurons (Khakh and Henderson, 1998).
Interestingly, Harris and Constanti (1995) have shown that ZD 7288 blocks IH when administered
intracellularly and that this blocking effect is more rapid (~2 min)
than when it is applied extracellularly (~15 min). Because the drug
appears to be partly lipophilic in physiological solutions, these
authors argued that ZD 7288 may block
IH channels by binding at a an
intracellular site (Harris and Constanti, 1995). In agreement with this
proposal, the blockade of IH in MNCs
by ZD 7288 (, ~15 min) was much slower than that caused by
Cs+ (, ~15 sec; our unpublished
results), which is known to block the channels at an extracellular site
(Harris and Constanti, 1995). The slow time course of action of ZD 7288 in MNCs, therefore, probably reflects the time required for the drug to
permeate the plasma membrane.
Similar to what has been found in neurons of the SNC (Harris and
Constanti, 1995) and hippocampus (Gasparini and DiFrancesco, 1997), the
blockade of IH by ZD 7288 in MNCs
could not be significantly reversed by washout periods lasting 1 hr
(p > 0.05; n = 3). In SNC
neurons, the block of IH by ZD 7288 was found to be significantly relieved by hyperpolarization, manifested
by the appearance of a slowly developing inward current while the cell
was held at 100 mV (, ~25 sec; Harris and Constanti, 1995).
Because blockade of IH was not
use-dependent, the authors concluded that the affinity of the binding
site for ZD 7288 can be reduced as a result of a conformational change
that takes place during membrane hyperpolarization. Surprisingly, we
could not significantly reverse the ZD 7288-induced block of
IH in MNCs, even by applying large
hyperpolarizing steps (e.g., to 115 mV) for periods lasting up to 15 min (data not shown). The molecular structure of the channels
underlying IH in MNCs, therefore, may
be different than those present in SNC neurons. Future studies will be
required to identify which of the genes encoding
IH channel subunits (Ludwig et al.,
1998; Biel et al., 1999; Ishii et al., 1999) are expressed in these
different types of neuron.
Role of IH in the control of
electrical activity
Bath application of ZD 7288 consistently evoked hyperpolarizations
when the initial membrane potential of MNCs was less than 65 mV. The
amplitude of this effect was proportional to the initial voltage of the
cells (data not shown), in agreement with the suppression of an
increasing density of active IH at
progressively more negative potentials. Application of ZD 7288 to cells
held at potentials between 65 mV and spike threshold (approximately
52 mV), however, did not have significant effects on membrane
potential (Table 2). This finding implies that in silent MNCs resting
within ~10 mV of spike threshold, IH
is largely inactive and, therefore, might not significantly contribute
to excitability. Surprisingly, bath application of ZD 7288 produced
robust inhibitory effects on both phasic and continuous firing recorded
from spontaneously active MNCs (Figs. 7-9). These effects were not
attributable to a change in synaptic activation or to differences in
Ca2+-dependent afterpotentials (Table 2).
The inhibition of MNCs by ZD 7288, therefore, was presumably
attributable to the hyperpolarization that resulted from the blockade
of active IH. This conclusion implies
that the presence of spiking activity can somehow lead to the
activation of GH and to the production
of an IH-dependent excitatory drive.
How does the presence of spiking activity trigger the activation of
IH? In phasically active cells, the
afterhyperpolarization (AHP) that follows individual bursts might serve
as a stimulus for the voltage-dependent activation of
IH, as occurs in thalamic neurons
(McCormick and Pape, 1990; Pape, 1996). However, the slow depolarizing
phase that precedes the onset of a subsequent burst normally lasts
several tens of seconds. Because any
IH activated by the postburst AHP
would presumably deactivate during this period, it seems unlikely that
such a mechanism could underlie the excitatory role of
IH during phasic firing in rat MNCs.
Another possibility is that the transient membrane hyperpolarizations
produced by consecutive HAPs can lead to the cumulative activation of a
sufficiently large IH to contribute a
measurable excitatory drive during spontaneous firing. We tested this
hypothesis by examining the effects of trains of brief (2-4 msec)
hyperpolarizing pulses (100-600 pA) delivered at frequencies of 5-10
Hz. The response of MNCs to such pulses consisted of a rapidly rising
negative phase followed by an exponential recovery of the membrane
potential (, ~15 msec), thus approximating the shape and amplitude
of postspike HAPs (Bourque et al., 1985). As illustrated in Figure
10, such trains readily provoked firing
in MNCs held at voltages near threshold. Moreover, in each of three
cells tested, bath-application of 30-60 µM ZD 7288 progressively blocked this effect with a time course consistent with the effects of the drug on IH
(Figs. 1, 2). These findings suggest that HAPs may indeed mediate the
activation of IH during periods of
action potential firing. Finally, another possibility is that
Ca2+ influx during action potentials, or
perhaps during the rebound phase of each HAP, can lead to a reversible
activation of IH. In thalamic neurons,
for example, transient increases in intracellular [Ca2+] appear to cause a reversible
augmentation of IH that is
attributable to the rapid, Ca2+-dependent,
formation of cyclic nucleotides (Luthi and McCormick, 1999). An
activity-dependent production of cyclic nucleotides in MNCs might
therefore be responsible for the appearance of
IH at subthreshold voltages. Further
studies will be required to determine if this mechanism contributes an
excitatory drive during spontaneous firing.
View larger version (28K):
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Figure 10.
Repetitive negative transients generate ZD
7288-sensitive excitation. Trains comprising 52 pulses (3 msec each)
were delivered at a rate of 1 per 130 msec (~7.7 Hz;
top) in a single cell. The voltage response to each
pulse closely resembled post-spike HAPs (see Discussion). In the
absence of ZD 7288 (control) trains of pulses of varying amplitude
could readily evoke firing. Bath application of 60 µM ZD
7288, however, caused the gradual disappearance of this response,
suggesting that trains of negative voltage transients can activate
IH.
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FOOTNOTES |
Received Nov. 9, 1999; revised March 13, 2000; accepted April 17, 2000.
This work was supported by an operating grant from the Medical Research
Council (MRC) of Canada to C.W.B. and by MRC Studentship and Senior
Scientist Awards to M.G.L. and C.W.B., respectively. We thank R. Buss
for expert assistance in the analysis of spontaneous synaptic events
and S.H.R. Oliet for critically reading this manuscript.
Correspondence should be addressed to Dr. Charles W. Bourque, Division
of Neurology, Room L7-216, Montreal General Hospital, 1650 Cedar
Avenue, Montreal, Quebec H3G 1A4, Canada. E-mail:
mdbq{at}musica.mcgill.ca.
| |
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ABSTRACT
INTRODUCTION
