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Calcium-Independent Afterdepolarization Regulated by Serotonin in
Anterior Thalamus
ESTHER M. CHAPIN AND RODRIGO ANDRADE
Department of Psychiatry and Behavioral Neurosciences and Cellular and Clinical Neurobiology Training Program,
Wayne State University School of Medicine, Detroit, Michigan 48201
INTRODUCTION
After a hyperpolarization, thalamic neurons can generate a
transient afterdepolarization (ADP) that plays a key role in
generating the interval between spindle oscillations in relay
thalamic nuclei such as the lateral geniculate nucleus (Bal and
McCormick 1996). Previous studies have shown that this ADP
reflects upregulation of the hyperpolarization-activated current
Ih (Bal and McCormick 1996) and suggested that the triggering
factor for this upregulation is an increase in intracellular calcium after low-threshold calcium spikes (Luthi and McCormick 1998).
Interestingly, anterior thalamic nuclei do not exhibit spindle
oscillations, although the electrophysiological properties of the
neurons comprising these nuclei appear to be similar to those
of better studied thalamic neurons such as those of the lateral
geniculate (Paré et al. 1987). We now report that in the anterodorsal nucleus of the thalamus (ADn), there is also an
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Ih-mediated ADP, but that a large component of the effect is
triggered calcium independently. Furthermore we find that
serotonin can enhance the inward current associated with this
ADP.
METHODS
Brain slices were prepared using standard protocols. Whole cell
recordings were obtained from the ADn using the blind-tight-seal
patch-clamp recording technique. The intracellular solutions are described in Table 1.
Recordings were obtained using an Axoclamp 2B amplifier (Axon
Instruments, Foster City, CA). Electrical signals were filtered at 10
kHz and recorded with a paper chart recorder (Model 3300, Gould
Instruments, Valley View, OH) or were digitized using a Digidata
1200 under the control of Axoclamp 7 (Axon Instruments). Voltageclamp experiments were conducted using the continuous voltageclamp mode of the amplifier. Recordings were considered acceptable
if the series resistance was ⬍30 M⍀ and could be compensated by
⬎70%. Magnitude of Ih was determined by subtracting the instantaneous current from the steady-state current. Drugs were administered
in the Ringer perfusing the bath. Most drugs were obtained from
Sigma (St. Louis, MO). Data was analyzed using Origin (Microcal
Software, Northampton, MA).
RESULTS
In our slices, neurons of the ADn are quiescent and exhibit
a resting membrane potential near ⫺65 mV. When transiently
hyperpolarized with current injection, these cells exhibit a slow
rebound afterdepolarization or ADP (Fig. 1A1). This is similar
to results obtained in other nuclei of the thalamus (Bal and
McCormick 1996), using the same stimulation protocol. In the
geniculate, the ADP has been attributed to calcium influx
secondary to rebound calcium spikes. Surprisingly, in the ADn,
a significant ADP remains (ⱖ50%, n ⫽ 3 cells, Fig. 1) even
after blocking the calcium spikes with cadmium and nickel. If
at least one component of the ADP in the ADn was calcium
independent, then a strong ADP would be detectable after a
single long hyperpolarization (Luthi and McCormick 1998).
As can be seen in Fig. 1B, ADn cells when stimulated using
this protocol, still express a robust ADP or, in voltage clamp,
a slow inward after current of similar time course (IADP).
Because these results suggested the presence of a calciumindependent ADP in the ADn, we next examined the ability of
hyperpolarizing pulses to elicit an ADP (or IADP) after buffering intracellular calcium with EGTA and blocking calcium
influx. Even under these conditions, a strong IADP was observed (n ⫽ 6 cells, Fig. 1C). Of course, one possible inter-
0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society
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Chapin, Esther M. and Rodrigo Andrade. Calcium-independent
afterdepolarization regulated by serotonin in anterior thalamus. J.
Neurophysiol. 83: 3173–3176, 2000. Previous studies have identified
an afterdepolarization (ADP) in thalamocortical neurons that is mediated by an upregulation of the hyperpolarization-activated current
Ih. This ADP has been suggested to play a key role in the generation
of spindle oscillations. In the lateral geniculate nucleus, upregulation
of Ih has been shown to be signaled by a rise in intracellular calcium
leading to the activation of adenylate cyclase and formation of cAMP.
However, it is unclear how generalizable this mechanism is to other
thalamic nuclei. We have used whole cell recording to examine the
electrophysiological properties of neurons of the anterodorsal thalamic nucleus, a nucleus thought not to undergo spindle oscillations.
We now report that cells in this nucleus also display an ADP mediated
by Ih. Surprisingly, the ADP and the underlying upregulation of Ih
persisted even after buffering intracellular calcium and blocking calcium influx. These results indicate that, in neurons of the anterodorsal
thalamic nucleus, an Ih-mediated ADP can occur through a mechanism that does not involve a rise in intracellular calcium. We next
examined the possibility that this calcium-independent ADP might be
modulated by serotonin. Serotonin produced a robust enhancement in
the amplitude of the ADP even after strong buffering of intracellular
calcium and blockade of calcium channels. These results indicate that
neurons of the anterodorsal thalamic nucleus display a calciumindependent, Ih-mediated ADP and that this ADP is a target for
regulation by serotonin. These findings identify a novel mechanism by
which serotonin can regulate neuronal excitability.
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TABLE
E. M. CHAPIN AND R. ANDRADE
1. Description of intracellular solutions
Intracellular
Solution
Potassium
Gluconate
Buffer
No buffer
10 mM EGTA
25 mM BAPTA
115
115
80
0.20 EGTA
10 EGTA
25 BAPTA
CaCl2
Estimated
[Ca2⫹]f, nM
GTP
HEPES
ATP
MgCl2
NaCl
—
100
100
0.5
0.5
0.5
10
10
10
2
2
2
2
2
2
5
5
5
—
1
7.5
All concentrations are in mM. BAPTA, bis(o-aminophenoxy)-N,N,N⬘,N⬘-tetraacetic acid; EGTA, Bis (b-aminoethyl ether)-N,N,N⬘,N⬘-tetraacetic acid; GTP
guanosine triphosphate; ATP, adenosine triphosphate.
Upregulation of Ih is responsible for the ADP
In other areas of the thalamus, the ADP triggered by hyperpolarizing current injections is mediated by Ih (Bal and Mc-
Cormick 1996; Luthi and McCormick 1998). Therefore we
tested whether this calcium-independent ADP is similarly due
to a facilitation of Ih. As illustrated in Fig. 1C3, administration
of 25–100 ␮M of the selective Ih inhibitor ZD7288 (BoSmith
et al. 1993) blocked the ADP and IADP in four cells. In the same
cells, ZD7288 also blocked Ih measured directly. These results
suggest that IADP is mediated by Ih. If this was the case, it
should be possible to directly observe an upregulation of Ih
after the hyperpolarizing pulse. As shown in Fig. 2A, Ih activated by a small hyperpolarizing step given every 20 s was
stable, displaying no detectable facilitation with pulse number.
However, if a large hyperpolarization (to ⫺100 mV) was
substituted for the test step, the magnitude of Ih activated on
the subsequent test steps was significantly increased (n ⫽ 3
cells, P ⬍ 0.01). This facilitation decayed during 1–2 min with
a time course that is well fitted by a single exponential (␶ ⫽
27.34 ⫾ 4.00 s).
These experiments were conducted using 10 mM EGTA.
Therefore to ascertain that this was not due to any residual
calcium transient, we repeated this experiment using 25 mM
BAPTA in the intracellular solution while bathing the cells
with 3 mM nickel and 200 ␮M cadmium. As shown in Fig. 2B,
FIG. 1. Properties of the afterdepolarizing potential
(ADP) in the anterior nucleus of the thalamus (ADn).
A1: 20 hyperpolarizing pulses at 20 Hz with 120-ms
duration induces an ADP at rest. A2: in presence of
calcium channel blockers 3 mM nickel (Ni) and 200
␮M cadmium (Cd), the hyperpolarizing pulses induce
an ADP ⬃66% of control. Vm ⫽ ⫺60 mV. Calibration
bar: 5 mV and 20 s. A3: superimposed records of ADP
from A, 1 and 2. B1: hyperpolarizing pulse induces an
ADP at rest. Vm ⫽ ⫺60 mV. B2: current underlying the
ADP (IADP) recorded in voltage-clamp mode in the
same cell. Vh ⫽ ⫺60 mV. Calibration bar: 2 mV, 50
pA, and 10 s. Cells in A and B were recorded using a
low-EGTA intracellular solution. C: calcium-independent form of IADP. C1: administration of 3 mM Ni and
200 ␮M Cd blocks the rebound calcium spike after an
800-ms-long hyperpolarizing pulse. Vm ⫽ ⫺60 mV. //,
lapse in time. C2: robust IADP still can be recorded after
buffering intracellular calcium with 10 mM EGTA and
blocking calcium channels with 3 mM Ni and 200 ␮M
Cd. C3: in the same cell, 100 ␮M ZD7288 blocks the
ADP. Calibration bar: 25 pA and 10 s.
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pretation for these results may be that residual calcium influx
could be responsible for the ADP. However, administration of
cadmium and nickel completely blocked the rebound calcium
spike (Fig. 1C1), and 10 mM EGTA should be adequate to
clamp calcium transients (Neher 1988). Nevertheless, because
the kinetics of calcium chelation by EGTA is slow, it remained
possible that a small residual influx of calcium might have
produced a transient local calcium rise. Therefore we repeated
the previous experiment using 25 mM bis-(o-aminophenoxy)N,N,N⬘,N⬘-tetraacetic acid (BAPTA) in the intracellular solution, a manipulation that should allow for faster calcium chelation and produce supramaximal calcium buffering capacity
(Neher 1988). In seven cells tested using this procedure, we
consistently observed a robust IADP even after extensive intracellular exchange of the cytoplasm with the recording solution
(⬎10 min). These results strongly suggest that a rise in intracellular calcium concentration is not necessary for the triggering of IADP in the ADn.
SEROTONIN REGULATES A CALCIUM-INDEPENDENT ADP
even under these conditions, the facilitation of Ih remained
unimpaired. In fact, the time constant for the decay of the
facilitation was essentially identical to that observed with 10
mM EGTA (␶ ⫽ 27.75 ⫾ 3.92 s). These observations strongly
suggest that hyperpolarization can facilitate Ih in the ADn and
that this facilitation is independent of changes in intracellular
calcium concentration.
Effect of serotonin on the hyperpolarization-induced Ih
facilitation
Serotonin is known to modulate Ih (Bobker and Williams
1990). Therefore we asked whether serotonin would also facilitate the ADP. As illustrated in Fig. 3, serotonin (10 ␮M)
significantly increased the IADP (n ⫽ 10 cells, Fig. 3A). This
increase reflected a genuine increase in the upregulation of Ih
because ZD7288 blocked IADP in control and in the presence of
serotonin (n ⫽ 4 cells, data not shown). To determine if this
effect reflects an increase in the calcium-independent form of
the IADP, we repeated these experiments while perfusing the
slices with the calcium channel blockers cadmium (200 ␮M)
and nickel (2 mM) and buffering intracellular calcium with 10
mM EGTA (n ⫽ 2 cells) or 25 mM BAPTA (n ⫽ 2 cells). As
illustrated in Fig. 3B, even under these conditions, serotonin
was still capable of inducing a strong facilitation of the IADP.
DISCUSSION
In the present study we have found that cells in the ADn
generate an Ih-mediated ADP and IADP in response to hyper-
polarization and that there is a significant portion of the ADP
that does not depend on calcium influx. Whereas previous
studies found that intracellular calcium chelation could block
the ADP (Luthi and McCormick 1998), we found that a strong
ADP survived even after buffering intracellular calcium and
blocking calcium channels. Further, we found that serotonin
could increase this calcium independent form of the IADP.
How can a hyperpolarization induce this ADP? Luthi and
McCormick (1999) have proposed a key role for cAMP in the
generation of the ADP. In the geniculate, calcium entering
during the rebound calcium spike indirectly facilitates Ih by
activating adenylate cyclase. Because cAMP shifts the voltage
dependence of Ih toward more depolarized voltages, the net
effect of the calcium influx is to upregulate Ih, an effect that is
seen as an ADP at resting membrane voltages. A similar
mechanism could account for the calcium-independent ADP
we describe herein. It is known that the transition from the
closed to the open state of the Ih channel is associated with
increased affinity for cAMP (DiFrancesco 1999; Luthi and
McCormick 1998). Thus hyperpolarization, by opening Ih
channels and increasing their affinity for cAMP, could produce
the calcium-independent IADP if sufficiently high levels of
cAMP were available inside ADn neurons. Because biophysical studies suggest that the cAMP affinity for the open Ih
channel is in the nanomolar range (⬃70 nM) (Di Francesco
1999), this possibility does not seem out of the question.
Serotonin can shift the voltage dependence of Ih by a cAMPdependent mechanism (Bobker and Williams 1990). A similar
effect occurs in the ADn (unpublished data). If the ADP in the
ADn occurred through the mechanism outlined above, then one
could expect significant facilitation of the ADP by serotonin.
This is precisely what we observed. In fact, calcium spikes also
can potentiate the ADP through a calcium-induced increase in
cAMP (Luthi and McCormick 1999). Serotonin and an increase in intracellular calcium, therefore could work in similar
ways to facilitate the ADP. Of course, further studies will be
required to determine whether this, or another mechanism,
accounts for the ability of serotonin to facilitate the ADP.
What is the function of the ADP in the ADn? The ADP in
thalamic neurons plays an important role in generating the
interval between spindle oscillations. However, cells of the
anterior thalamus, including the ADn, are thought not to undergo spindle oscillations in the cat or the rat. Although this
initially was thought to result from the lack of a GABAergic
input from the reticular nucleus (Paré et al. 1987), recent
FIG. 3. Serotonin facilitates IADP. A: summary plot illustrating the effect of
serotonin (10 ␮M) on the ADP. Cells were recorded using 10 mM EGTA and the
ADP was induced by hyperpolarizing the cells using a slow ramp. *P ⬍ 0.05 in
t-test from average IADP values. B: effect of serotonin on the ADP in a cell
recorded using 10 mM EGTA and bathed in 3 mM Ni and 200 ␮M Cd. In this cell,
the ADP was triggered using 2 hyperpolarizing steps. Vh ⫽ ⫺60 mV.
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FIG. 2. Ih is facilitated by a strong hyperpolarization. A: facilitation of Ih by
hyperpolarization observed using 10 mM EGTA to buffer intracellular calcium. Cells were held at ⫺40 mV, and Ih was measured by stepping to ⫺75
mV for 4 s (inset). Facilitation was induced by replacing the test voltage step
for one to ⫺100 mV. B: facilitation observed when 25 mM BAPTA was used
to buffer intracellular calcium and cells were bathed in 3 mM Ni plus 200 ␮M
Cd, n ⫽ 3 cells. *P ⬍ 0.05 in t-test from average baseline values. Average of
the 3 baseline measurements was used to normalize the currents for each cell.
Lines reflect best fit using a single exponential.
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E. M. CHAPIN AND R. ANDRADE
studies have documented a significant input from this nucleus
to the anterior thalamus (Pinault and Deschenes 1998). This
raises the possibility that differences in the electrophysiological properties of neurons in the anterior thalamus might contribute to the unique network properties of this region. Here we
have shown that the cells in the ADn express a prominent ADP
that can be triggered solely by membrane hyperpolarization
and greatly enhanced by the neuromodulator serotonin. Further
studies will be required to determine how generally applicable
these results are to other nuclei of the thalamus and how these
features contribute to the firing of ADn neurons.
Received 11 November 1999; accepted in final form 27 January 2000.
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We thank Dr. William Spain for helpful comments on the manuscript.
This work was supported by National Institute of Mental Health Grant
MH-43985.
Address for reprint requests: E. M. Chapin, Dept. of Psychiatry and Behavioral Neurosciences, 2309 Scott Hall, Wayne State University School of
Medicine, 540 E. Canfield, Detroit, MI 48201.
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