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


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INTRODUCTION
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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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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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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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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Table 1. Description of intracellular solutions

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 MOmega 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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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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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 µM 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 µM 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 µM Cd. C3: in the same cell, 100 µM ZD7288 blocks the ADP. Calibration bar: 25 pA and 10 s.

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 (tau  = 27.34 ± 4.00 s).



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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 µM 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.

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 (tau  = 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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Fig. 3. Serotonin facilitates IADP. A: summary plot illustrating the effect of serotonin (10 µM) 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 µM Cd. In this cell, the ADP was triggered using 2 hyperpolarizing steps. Vh = -60 mV.


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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.


    ACKNOWLEDGMENTS

We thank Dr. William Spain for helpful comments on the manuscript.

This work was supported by National Institute of Mental Health Grant MH-43985.


    FOOTNOTES

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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