Department of Psychiatry and Behavioral Neurosciences and Cellular and Clinical Neurobiology Training Program, Wayne State University School of Medicine, Detroit, Michigan 48201
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ABSTRACT |
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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 calcium-independent, 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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INTRODUCTION |
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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
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.
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METHODS |
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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.
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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). Voltage-clamp
experiments were conducted using the continuous voltage-clamp 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).
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RESULTS |
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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 (
50%, 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).
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Because these results suggested the presence of a calcium-independent
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 interpretation
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.
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 McCormick 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 µM 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).
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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 µM cadmium. As shown in Fig. 2B, 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 µM) 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 µM) 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.
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DISCUSSION |
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In the present study we have found that cells in the ADn generate
an Ih-mediated ADP and
IADP in response to hyperpolarization 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 cAMP-dependent 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 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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 November 1999; accepted in final form 27 January 2000.
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REFERENCES |
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